Document reY8Xje76Z3YaDeY7qJ6d3qzV

TC RESEARCH TOXICOLOGY CENTRE - ROMA IIIMIM7850 BACTERIAL MUTATION ASSAY (S. typhimurium and E. cola) FINAL REPORT RTC Study no.: 52400 Sponsor: SOLVAY SOLEXIS S.p.A. V iale Lombardia, 20 20021 Bollate (MI) Italy Total number of pages: 58 RTC S.p.A. Via Tito Speri, 12 00040 Pomezia (Roma) - ITALY Tel.: +39.06.91095.1 Fax: +39.06.910.5737 e-mail: Mrtc.it www.rtc.it RTC S.p.A. Via Tito Speri, 12 00040 Pomezia (Roma) - ITALY Tel.: + 39.06.91095.1 Fax: + 39.06.912.2233 P.O. Box 15301-00143 - Roma Eur Laurentino RTC S.p.A. Capitale aociale Euro 5.164.000 C.C.I.A.A. n' 375376 Reg. Soc. Trib. di Rome ri* 2828/72 Cod. Fisc.: 00653120584 Partita IVA: 00920611001 7850 BACTERIAL MUTATION ASSAY (S. typhimurium and E. col,) RTC Study no.: 52400 FINAL REPORT I, the undersigned, was responsible for the preparation of this report. Date RTC Study No.: 52400 Page2 COMPLIANCE STATEMENT We, the undersigned, hereby declare that the following report constitutes a true and faithful account of the procedures adopted, and the results obtained in the performance of the study. The aspects of the study conducted by Research Toxicology Centre S.p.A. were performed in accordance with: A. Good laboratory practice for non clinical laboratory studies, U.S. Food and Drug Administration, Code of Federal Regulations, 21 Part 58, 22 December 1978 and subsequent revisions. B. Decreto Legislativo 27 Gennaio 1992 n. 120, Adoption of88/320/EEC and 90/18/EEC Directives on the inspection and verification of good laboratory practice (G. U. 18 Febbraio 1992 n. 40) and subsequent revisions. C. Directive 2004/10/EC of European Parliament and of the Council of 11 February 2004. On the harmonisation of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances. D. ENV/MC/CHEM(98) 17 OECD principles on Good Laboratory Practice (as revised in 1997). RTC Study No.: 52400 Date Page3 QUALITY ASSURANCE STATEMENT (Relevant to those aspects of the study conducted by RTC S.p.A.) Study phases monitored by RTC's QAU According to current relevant Standard Operating Procedures PROTOCOL CHECK Quality Assurance Inspections a Month Year Inspection Report to Report to Study Company Director Management 06.02.2006 07.02.2006 07.02.2006 PROCESS-BASED INSPECTIONS RELATED TO TIDS TYPE OF STUDY Dose preparation Treatment Plating out Plate scoring 04.05.2006 17.01.2006 07.02.2006 03.03.2006 11.05.2006 31.01.2006 27.03.2006 09.03.2006 Other process-based inspections were carried out on routine activities not directly related to this type of study. The relevant documentation is kept on file although specific inspection dates are not reported here. Associated laboratories and su ort functions are subect to re lar facili ins ections. FINAL REPORT Review completed Review of this report by RTC's QAU found the reported methods and procedures to describe those used and the results to constitute an accurate r resentation of the recorded raw data. RTC Study No.: 52400 Page4 Contents Page 1. SUMMARY ................................................................................................................................7 2. INTRODUCTION .......................................................................................................................9 2.1 Purpose..............................................................................................................................9 2.2 Principles of the method....................................................................................................9 2.3 Study organisation.............................................................................................................9 3. MATERIAL S AN D METHODS ..............................................................................................11 3.1 Test item..........................................................................................................................11 3.2 Control items...................................................................................................................11 3.3 Media ..............................................................................................................................12 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............................................................................................................14 3.6.3 Incubation andscoring ....................................................................................................14 4. RESULTS..................................................................................................................................15 4.1 Solubility test ..................................................................................................................15 4.2 Toxicity test.....................................................................................................................15 4.3 Assay for reverse mutation..............................................................................................15 5. AN AL YSIS OF RESULTS.......................................................................................................17 5.1 Criteria for outcome of the assays...................................................................................17 5.2 Evaluation .......................................................................................................................17 6. CON CL USION .........................................................................................................................18 7. KEYTOTABLES 1-20 ............................................................................................................19 7.1 Structure ofTables 3-20..................................................................................................19 7.2 Regression line ................................................................................................................19 Tables TABLE 1 -Toxicity test without metabolic activation .......................................................................20 TABLE 2 -Toxicity test with metabolic activation ............................................................................21 TABLE 3 -Experiment I - Plate incorporation method-TA1535......................................................22 TABLE 4 - Experiment I - Plate incorporation method - TAI 535 ......................................................23 TABLE 5 -Experiment I - Plate incorporation method-TA1537......................................................24 TABLE 6 -Experiment I - Plate incorporation method-TA1537......................................................25 TABLE 7 -Experiment I - Plate incorporation method- WP2uvrA...................................................26 TABLE 8 -Experiment I - Plate incorporation method- WP2uvrA...................................................27 TABLE 9 -Experiment I - Plate incorporation method-TA98..........................................................28 TABLE 10 -Experiment I - Plate incorporation method-TAl00......................................................29 TABLE 11 -Experiment II - Pre-incubation method-TA1535 .........................................................30 TABLE 12 -Experiment II - Pre-incubation method-TA1535 .........................................................31 TABLE 13 -Experiment II - Pre-incubation method-TA1537 .........................................................32 TABLE 14 -Experiment II - Pre-incubation method-TA1537 .........................................................33 TABLE 15 -Experiment II - Pre-incubation method- WP2uvrA.......................................................34 TABLE 16 -Experiment II - Pre-incubation method- WP2uvrA.......................................................35 TABLE 17 -Experiment II - Pre-incubation method-TA98 .............................................................36 RTC Study No.: 52400 Page5 Contents Page TABLE 18 - Experiment II- Pre-incubation method -TAl00 ...........................................................37 TABLE 19- Experiment ill- Plate incorporation method- WP2uvrA ..............................................38 TABLE 20 - Experiment III - Pre-incubation method - WP2uvrA .....................................................39 Appendices APPENDIX 1 - Historical control data without metabolic activation.................................................40 APPENDIX 2 - Historical control data with metabolic activation......................................................41 Addenda ADDENDUM I - Certificate of analysis .............................................................................................42 ADDENDUM II - Study Protocol .......................................................................................................45 RTC Study No.: 52400 Page6 1. SUMMARY 1.1 The test ite7850 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,TAIO0 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. Test item solutions were prepared using ethanol. 1.2 In the toxicity test,the test item was assayed at a maximum dose-level of 5000 g/plate and at four lower dose-levels: 1580,500,158 and 50.0 g/plate. In the absence of S9 metabolic activation,signs of toxicity were observed at higher dose-levels with TA1535,TA1537 and WP2uvrA tester strains and at the highest dose-level with TA98 and TAl00. In the presence of S9 metabolism, signs of toxicity were observed at the highest concentration tested with TA1535 and TAI00. Two main experiments were performed. In Main Assay I, using the plate incorporation method,treatments were performed using the following dose-levels: Tester strain TA1535 TA1537 WP2uvrA TA98, TAl00 TA1535, TAl00 TA1537,TA98,WP2uvrA S9 Dose-levels (g/plate) - 200,100,50.0,25.0,12.5,6.25 - 100,50.0,25.0,12.5,6.25, 3.13 - 50.0,25.0,12.5,6.25,3.13,1.56 5000,2500,1250,625,313,156 + + 5000,2500,1250,625,313 As no increases in revertant numbers were observed, all treatments of Main Assay II. included a pre-incubation step. Toxicity results obtained in the first experiment were taken into consideration selecting the dose-levels for Main Assay II. Concentrations tested were as follows: Tester strains TA1535 TA1537 WP2uvrA TA98, TAl00 TA1535, TA1537 TA98, TAl00,WP2uvrA S9 Dose levels (g/plate) - 200,100,50.0,25.0,12.5,6.25 - 100,50.0,25.0,12.5,6.25,3.13 - 50.0,25.0, 12.5,6.25,3.13,1.56 - 2500,1250,625,313,156,78.1 + 5000,2500,1250,625,313,156,78.1 + 5000,2500,1250,625,313,156 RTC Study No.: 52400 Page 7 No toxcity was observed in the absence of S9 metabolism with WP2uvrA tester strain using the plate inorporation or the pre-incubation method. In order to evaluate a potential mutagenic effect up to cytotoxic concentrations, an additional experiment was carried out with this tester strain using a maximum concentration of 800 g/plate and four lower dose-levels. Treatments were performed using both the plate incorporation and the pre incubation method. 1.3 The test item did not induce two fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assays, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism. 1.4 It is concluded that the test item 7850 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions. RTC Study No.: 52400 Page8 2. INTRODUCTION 2.1 Purpose This report describes experiments performed to assess the mutagenic activity of the test item to Salmonella typhimurium strainsTA1535,TA1537,TA98 andTAIO0, 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: EEC Council Directive 2000/32, Annex 4D. 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 2002 1 Bollate (Ml) Italy Location of Study: ResearchToxicology Centre S.p.A. Genetic Toxicology Department ViaTito Speri, 12 00040 Pomezia (Rome) Italy RTC Study No.: 52400 Page9 Principal dates: Study protocol approved by Study Director: 18-Jan-2006 Study commenced: 28-Feb-2006 (Toxicity assay treatment) Study completed: 17-Mar-2006 (Completion of scoring Main Assay ill) Archiving: The original data arising from this study and a copy of the final report consigned will be stored in the archives ofResearch Toxicology Centre S.p.A. for a period of3 years from the date of consignment of the report. At the completion of this period the Sponsor will be contacted for despatch or disposal of the material, or further archiving. An aliquot of the test item will be retained within the archives of the testing facility for a period of 10 years after which it will be destroyed. RTC Study No.: 52400 Page JO 3. MATERIALS AND METHODS 3.1 Test item Details of the test item received at RTC were as follows : Name Batch Purity Received from Date received Amount received Description Expiry date Container Storage at RTC RTC reference number 7850 3223 ON >75% (referred to carboxylic end groups) Solvay Solexis 20-Feb-2006 100 g colourless liquid 2012 opaque plastic bottle room temperature 1 0022 On 27-Feb-2006 a 12 g sub-sample of the test item was transferred from the Formulation Unit to the Department of Genetic and Cellular Toxicology and stored under the same conditions. Solutions of the test item, as received, were prepared immediately before use in ethanol. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 2 hours and 30 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 03H2802). Dimethylsulphoxide (DMSO) (Fluka AG, batch 1060564 41204083). Ethanol (Carlo Erba, batch 402973041) Positive control treatments used solutions prepared as follows: Sodium azide (Moltox, Inc., batch 6258SA) in distilled water. 9-Aminoacridine (Moltox, Inc., batch 6030AC) in DMSO. 2-Nitrofluorene (Moltox, Inc., batch 2188NF) in DMSO. 2-Arninoanthracene (Moltox, Inc., batch 6269AA and Sigma, batch 58F-3462) in DMSO. Methylmethanesulphonate (MMS) (Fluka AG, batch 3593 1 6/153696) in distilled water. RTC Study No. : 52400 Page 1 1 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 ofthe tester strains. Nutrient Agar: Oxoid Nutrient Broth No 2 (25g) and Difeo Bacto-agar (1 5g) 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% Difeo Bacto-agar in Vogel Bonner Medium E, with 2% Glucose, and poured into 9 cm plastic Petri dishes. Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difeo Bacto-agar + 0.5% NaCl in distilled water. Prior to use l O ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) were added to the top agar ( 1 00 ml). 3.4 S9 tissue homogenate One batch of S9 tissue homogenate (designated 2006/1) was used in this study and had the following characteristics: S9 Batch Protein content (mg/ml) Aminopyrine demethylase activity (Mfg liver/5 min, formaldehyde production) 2006/1 36.5 2.42 4.59 0. 15 The S9 tissue fraction was prepared from the livers of five young male Sprague-Dawley rats which had received prior treatment with phenobarbital and betanaphthoflavone to induce high levels of xenobiotic metabolising enzymes. The efficacy of the S9 tissue fraction was previously checked in an Ames test and produced acceptable responses with the indirect mutagens 2-aminoanthracene and benzo(a)pyrene, using S. typhimurium tester strain TAl 00. Induced Salmonella typhimurium revertants: TAl 00 2-Aminoanthracene ( 1 g/plate) Benzo( a)pyrene (2.5 g/plate) 1 557 197 5 1 4 27 RTC Study No. : 52400 Page 12 The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each I 0 ml): S9 tissue fraction NADP ( I 00 mM) G-6-P (100 mM) KCl (330 mM) MgC12 (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 3.5 Bacterial strains Four strains ofSalmonella typhimurium (TA1 535, TA1 537, TA98 and TAl 00) and a strain of Escherichia coli (WP2 uvrA) were used in this study. 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 pKM l 0 l 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. 3.6 Methods 3 .6.1 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 this test a wide range of dose-levels ofthe test item, set at half-log intervals, was 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. RTC Study No. : 52400 Page 13 3 .6.2 Main experiments Three 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. 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 and a treatment series of the third experiment were performed using the 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 and a treatment series of the third experiment were performed using the pre-incubation method. The components were added in tum to an empty test-tube: (i) Bacterial suspension (ii) Test item solution or DMSO or positive control solution (iii) S9 mix or phosphate buffer (pH 7.4, 0. 1 M) 0. 1 ml 0.01 ml 0.05 ml 0.5 ml The incubate was vortexed and placed at 3 7C for 30 minutes. Two ml of overlay agar were then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. 3 .6.3 Incubation and scoring The prepared plates were inverted and incubated for approximately 72 hours at 3 7C. After this period of incubation, the scoring was effected by counting the number of revertant colonies on each plate. RTC Study No. : 52400 Page 14 4. RESULTS 4.1 Solubility test As indicated by the Sponsor, the test item was found to be soluble in ethanol at a concentration of 500 mg/ml. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. Since 1 00 l of the test item solution are used in the preparation of each plate, this permitted a maximum concentration of 5000 g/plate to be used in the toxicity test (the upper limit to testing indicated by the Study Protocol). 4.2 Toxicity test The test item was assayed using the plate incorporation method, at a maximum dose-level of 5000 g/plate and at four lower dose-levels spaced at approximately half-log intervals: 1 5 80, 500, 1 5 8 and 50.0 g/plate. Results are presented in Tables 1 and 2. In the absence of S9 metabolic activation, toxicity, as indicated by lack of colony growth, thinning of the background lawn and reduction in revertant numbers, was observed at higher dose-levels with TA 1 535, TA 1 537 and WP2uvrA and at the highest dose-level with TA98 and TA 1 00 tester strains. In the presence of S9 metabolism, toxicity was observed only at the highest concentration tested with TA 1 53 5 and TA I O0 tester strains. 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 20. In Main Assay I, using the plate incorporation method, the test items were assayed at the following dose-levels: Tester strain TA 1 535 TA 1 537 WP2uvrA TA98, TA l 00 TA 1 535, TA l 00 TA 1 53 7, TA98, WP2uvrA S9 Dose-levels (g/plate) - 200, 1 00, 50.0, 25.0, 12.5, 6.25 - 100, 50.0, 25.0, 12.5, 6.25, 3.13 - 50.0, 25.0, 12.5, 6.25, 3.13, 1.56 - 5000, 2500, 1 250, 625, 3 1 3, 1 56 + + 5000, 2500, 1250, 625, 3 1 3 Following treatment with 7850 in the absence of S9 metabolic activation, slight toxicity, as indicated by thinning of the background lawn, was observed at the highest concentration tested with TA 1 535 and TA 1 537 tester strains. With TA 98 and TA l 00 tester strains, reduction in revertant numbers was observed at 2500 and 5000 g/plate, while no relevant toxicity was observed with WP2uvrA. RTC Study No. : 52400 Page 15 Treatments in the presence of S9 metabolic activation showed thinning of the background lawn and/or reduction in revertant numbers at the highest concentration tested (5000 g/plate). As no increases in revertant numbers were observed, all treatments of Main Assay II included a pre-incubation step. Toxicity results obtained in the first experiment were taken into consideration selecting dose-levels for Main Assay II. Concentrations tested were as follow: Tester strains TA1 535 TA1 537 WP2uvrA TA98, TAl 00 TA1 535, TA1 537 TA98, TAl 00, WP2uvrA S9 Dose levels (g/plate) - 200, 100, 50.0, 25.0, 12.5, 6.25 - 1 00, 50.0, 25.0, 12.5, 6.25, 3 . 1 3 - 50.0, 25.0, 12.5, 6.25, 3 .1 3, 1 .56 - 2500, 1 250, 625, 3 1 3, 1 56, 78. 1 + 5000, 2500, 1250, 625, 3 1 3, 1 56, 78. 1 + 5000, 2500, 1 250, 625, 3 1 3, 1 56 In the absence of S9 metabolism, toxicity as indicated by thinning of the background lawn and reduction in revertant numbers was observed at the highest dose level tested with TA1 535, TA1 537 and TA98 tester strains. Signs of toxicity were also observed with TAIO0 over a concentration range from 625 to 2500 g/plate. In the presence of S9 metabolism toxicity, as indicated by thinning of the background lawn and reduction in revertant colonies, was observed at the three highest dose levels with all tester strains. No sign of toxicity was observed in the absence of S9 metabolism with WP2uvrA tester strain using the plate inorporation or the pre-incubation method. In order to evaluate a potential mutagenic effect up to cytotoxic concentrations, an additional experiment was carried out with this tester strain using a maximum concentration of 800 g/plate and four lower dose-levels spaced by a factor of two. Treatments were performed using both the plate incorporation and the pre-incubation method. No visible precipitate was observed in any experiment, at the end of the incubation period even at the highest concentration tested. No treatments with the test item produced increases in revertant numbers in the plate incorporation or pre-incubation assay, in any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. In Main Assay ill, sterility data of test item solutions, used for plate incorporation treatment, were not recorded. However, no contamination was observed in any plate treated with the test item at any dose level. Hence, this deviation to the Study Protocol was not considered to have affected the integrity of the study. 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. : 52400 Page 16 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 showin g increasing numbers of mutant colonies with increasing dose-levels. 5.2 Evaluation 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 7850 is not mutagenic to S. typhimurium and E. coli under the reported experimental conditions. RTC Study No. : 52400 Page 1 7 6. CONCLUSION It is concluded that the test item-7850 does not induce reverse mutation in Salmonella typhimurium or Esch the reported experimental conditions. RTC Study No. : 52400 Page 18 7. KEY TO TABLES 1-20 7.1 Structure ofTables 3-20 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 receive no treatment, while the 0.00 dose-level is the solvent control. 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 does not include the untreated control data, but includes the solvent 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 ofstudents "t" statistic, and the p-value for the regression lines are also given. RTC Study No. : 52400 Page 19 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i } TABLE 1 - Toxic i ty te s t without metabo l i c act ivation STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol EXPERIMENT : Toxic ity test Dose - l eve l ( g/plate } TA- 1 5 3 5 Rev/pl . TA- 1 5 3 7 Rev/pl . TA- 9 8 Rev/pl . TA- 1 0 0 Rev/pl . WP2 uvrA Rev/pl . Unt re a t e d 0 . 00 50. 0 158 500 1580 5000 16 21 28 12 0 25 19 17 33 118 30 15 19 36 123 18 9 0 * 32 134 15 10 0 * 39 126 21 9 0 * 38 111 21 6 0 * 16 70 19 * : Thinning of the background lawn RTC Study No. : 52400 Page 20 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i } TABLE 2 - Toxi c i ty t e s t with met abol i c act ivat ion STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol EXPERIMENT : Toxicity test Dos e - level ( g/p l at e } TA- 1 5 3 5 Rev/pl . TA- 1 5 3 7 Rev/pl . TA- 9 8 Rev/p l . TA- 1 0 0 Rev/pl . WP2 uvrA Rev/pl . Unt r e a t e d 0 . 00 so . a 158 500 1580 5000 17 24 44 132 31 21 19 46 110 28 17 25 34 116 32 15 24 43 118 32 12 24 47 117 34 16 19 46 12 0 28 8 * 19 37 94 29 * : Thinning of the background l awn RTC Study No. : 52400 Page 21 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 3 - Experiment I - Plate incorporation method - TA1 5 3 5 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol Stra i n : TA1 5 3 5 Dose - level [g/pl ] Without metabolic activation Plate counts Mean s . E . Titre : 222 Untreated 0 . 00 6 . 25 12 . 5 25 . 0 50. 0 100 200 18 17 14 16 1 . 2 15 18 20 18 1 . 5 14 13 10 12 1 . 2 10 13 16 13 1 . 7 15 15 11 14 1 . 3 13 11 16 13 1 . 5 16 8 14 13 2 . 4 10 * 10 * 13 * 11 1 . 0 Regression analys is : Points S9 Intercept 1-3 1 -4 1 1 - - 5 6 1-7 4 . 066 3 . 899 3 . 820 3 . 789 3 . 773 S lope -0 . 0485 - 0 . 0142 -0 . 0052 -0 . 0031 -0 . 0024 Corr . coeff . -0 . 59240 - 0 . 3 3 94 1 -0 . 24698 -0 . 26109 -0 . 40482 t 1 . 9455 1 . 1410 0 . 9190 1. 0819 1 . 9298 P -value 0 . 09278 0 .28045 0 . 37485 0 . 29533 0 . 06870 Positive and negat ive controls Treatment S9 Plate counts Un t r e a t e d Sodium Az ide 1 g/pl 18 17 14 478 470 550 Mean 16 499 s. E. 1.2 25 .4 * : Thinning of the background l awn RTC Study No. : 52400 Page 22 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 4 - Experiment I - Plate i ncorporation method - TA1 5 3 5 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : TA1 5 3 5 Dose - l evel [g/pl ] Titre : 2 2 2 With metabo l i c act ivation Plate counts Mean s . E . Unt r e a t e d 0 . 00 156 3 13 625 1250 2500 5000 16 13 14 14 0 . 9 19 15 19 18 1 . 3 12 17 16 15 1 . 5 14 18 17 16 1.2 18 12 16 15 1 . 8 10 10 15 12 1 . 7 12 9 14 12 1 . 5 7* 5* 7* 6 0.7 Regress ion analys i s : Points S9 Intercept 1-3 + 1-4 + 1-5 + 1-6 + 1-7 + 4 . 112 4 . 090 4 . 138 4 . 046 4 . 047 S l ope -0 . 0005 -0 . 0003 -0 . 0006 -0 . 0003 -0 . 0003 Corr . coef f . -0 .23377 -0 .25670 -0 . 62840 -0 . 62943 -0 . 85711 t 0 . 6361 0 . 8399 2 . 9127 3 . 2400 7 .2527 P - va l u e 0 . 54493 0 .42060 0 . 01211 0 . 00513 0 . 00000 Pos itive and negative control s Treatment S9 DMSO 2 -Aminoanthracene 100 1/pl + 1 g/pl + * : Tinning of the background l awn Plate counts 17 17 20 141 131 156 Mean 18 14 3 s. E. 1.0 7.3 RTC Study No. : 52400 Page 23 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 5 - Experiment I - Plate incorporation method - TA1 5 3 7 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : TA1 5 3 7 Dose - level [g/pl] Wi thout metabo l i c act ivat ion Plate count s Mean s . E . Titre : 2 2 0 Unt r e a t e d 0 . 00 3 . 13 6 . 25 12 . 5 25 . 0 50 . 0 100 16 16 19 17 1 . 0 12 12 11 12 0 . 3 13 14 12 13 0 . 6 13 13 15 14 0 . 7 13 12 15 13 0 . 9 11 11 12 11 0 . 3 11 12 7 10 1 . 5 14 * 14 * 13 * 14 0 . 3 Regres sion analys i s : Points S9 Intercept 1 -3 1 -4 1 -5 1-6 1-7 3 .431 3 . 500 3 . 596 3 . 622 3 . 510 S l ope 0 . 0448 0 . 0165 -0 . 0054 -0 . 0089 0 . 0000 Corr . coeff . 0 . 72587 0 . 46596 - 0 . 27144 -0 . 58170 -0 . 00110 t 2 . 7921 1 . 6653 1 . 0168 2 . 8606 0 . 0048 P - va l ue 0 . 02683 0 . 12682 0 . 32778 0 . 01133 0 . 99622 Pos itive and negat ive contro l s Treatment S9 Pl ate count s DMSO 9 -Aminoacridine 100 1/pl 50 g/pl 11 14 13 168 141 201 Mean 13 170 S. E. 0.9 17 . 3 * : Thinning of the background l awn RTC Study No. : 52400 Page 24 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 6 - Experiment I - Plate i ncorporation method - TA1 5 3 7 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : TA1 5 3 7 Dose - level [g/pl ] Titre : 2 2 0 With metabo l i c act ivat ion Plate count s Mean s . E . Unt r e a t e d 0 . 00 3 13 625 1250 2500 5000 16 18 19 10 14 15 13 12 17 13 9 16 12 12 11 10 13 8 7 7 6 18 0 . 9 13 1 . 5 14 1 . 5 13 2 . 0 12 0 . 3 10 1 . 5 7 0.3 Regre s s ion analys i s : Points S9 Intercept 1-3 + 1-4 + 1-5 + 1-6 + 3 . 648 3 . 672 3 . 672 3 . 693 S l ope -0 . 0001 -0 . 0002 -0 . 0002 -0 . 0002 Corr . coeff . -0 . 06665 -0 .27600 -0 .47809 -0 . 79504 t 0 . 1767 0 . 9080 1 . 9626 5 . 2430 P - va l u e 0 . 86472 0 . 38521 0 . 0714 6 0 . 00008 Pos i t ive and negat ive contro l s Treatment S9 Plate count s DMSO 2 -Aminoanthracene 100 1/pl + 16 15 18 1 g/pl + 106 92 104 Mean 16 101 S. E. 0.9 4.4 RTC Study No. : 52400 Page 25 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 7 - Experiment I - Plate incorporation method - WP2 uvrA STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : WP2 uvrA Dos e - l eve l [ g/pl ] Without metabol i c act ivat ion Plate count s Mean s . E . Unt r e a t e d 0 . 00 1 . 56 3 . 13 6 . 25 12 . 5 25 . 0 50 . 0 26 28 24 24 22 21 20 27 22 25 28 27 25 27 23 20 26 24 19 26 27 14 18 23 26 1.2 22 0 . 9 23 2 . 1 27 0 . 9 25 1 . 2 23 1 . 8 24 2 . 5 18 2 . 6 Titre : 3 0 0 Regre s s ion analys i s : Points S9 Intercept 1-3 1 -4 1-5 1 1 - - 6 7 4 . 672 4 . 782 4 . 883 4 . 895 4 . 970 S l ope 0 . 1402 0 . 04 96 0 .0033 0 . 0002 -0 . 0117 Corr . coeff . 0 . 63811 0 . 44022 0 . 05588 0 . 00602 -0 . 51111 t 2 . 1927 1 . 5504 0 .2018 0 . 0241 2 . 5920 P -value 0 . 06442 0 . 15208 0 . 84319 0 . 98108 0 . 01789 Pos itive and negative control s T r e a tme n t S9 Plate count s Unt r e a t e d MMS 5 00 g/pl 26 28 24 142 153 165 Mean 26 153 s. E. 1.2 6.6 RTC Study No. : 52400 Page 26 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE a - Experiment I - Plate incorporat ion method - WP2 uvrA STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : WP2 uvrA Dose - l eve l [g/pl ] Unt r e a t e d 0 . 00 313 625 12 5 0 2500 5000 Titre : 3 0 0 With metabo l i c act ivat ion Plate counts Mean s . E . 31 34 30 32 1 . 2 30 31 28 30 0 . 9 42 33 30 35 3 . 6 32 40 34 35 2 .4 26 25 31 27 1 . 9 30 31 29 30 0 . 6 33 * 30 * 27 * 30 1 . 7 Regres s ion analys i s : Points S 9 Intercept 1-3 + 1-4 + 1-5 + 1-6 + 5 . 515 5 . 771 5 . 707 5 . 656 S l ope 0 . 0008 -0 . 0003 -0 . 0001 0 . 0000 Corr . coeff . 0 . 53479 -0 . 29055 -0 . 27263 -0 . 23537 t 1 . 6745 0 . 9602 1 . 0217 0 . 9687 P - va l ue 0 . 13795 0 .35959 0 .32556 0 . 34712 Pos it ive and negat ive cont ro l s Treatment S9 Pl ate count s DMSO 2 -Aminoanthracene 100 1/pl + 10 g/pl + 29 23 31 179 146 189 Mean 28 171 S. E. 2 .4 13 . 0 * : Thinning of the background l awn RTC Study No. : 52400 Page 2 7 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 9 - Experiment I - Plate incorporat ion me thod - TA9 8 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol Strain : TA9 8 Dose - l eve l [g/pl ] Without metabo l i c act ivation Plate counts Mean s . E . Unt reated 0 . 00 156 313 625 1250 2500 5000 34 32 27 39 37 34 34 36 45 34 34 40 30 36 40 30 28 24 9 8 18 10 11 15 31 2 . 1 37 1 . 5 38 3 .4 36 2 . 0 35 2 . 9 27 1. 8 12 3 . 2 12 1 . 5 Titre : 2 5 1 With metabolic activation Plate counts Mean s . E . 34 35 44 43 49 47 NT NT NT 38 3 . 2 46 1.8 45 44 38 42 2 . 2 48 46 43 46 1 . 5 36 34 27 32 2 . 7 40 42 42 41 0 . 7 26 * 28 * 21 * 25 2 . 1 Regre s s ion analys i s : Point s S 9 Intercept 1-3 1 -4 1 1 - - 5 6 1-7 1 1 - - 3 4 + + 1 1 - - 5 6 + + 6 . 105 6 . 116 6 . 212 6 . 365 6 . 049 6 . 712 6 . 900 6 . 610 6. 707 S l ope -0 . 0002 -0 . 0003 -0 . 0007 -0 . 0011 -0 . 0006 -0 . 0001 -0 . 0009 -0 . 0002 -0 . 0003 Corr . coeff . - 0 . 08244 -0 . 20636 -0 . 71504 -0 . 91505 -0 . 86324 -0 . 08399 -0 . 76511 -0 . 36138 -0 . 78730 t 0 . 2189 0 . 6669 3 . 6879 9 . 0745 7 .4542 0 . 2230 3 . 7576 1 . 3974 5 . 1076 P -value 0 . 83301 0 . 51991 0 . 00273 0 . 00000 0 . 00000 0 . 82990 0 . 00374 0 . 18568 0 . 00011 Pos itive and negative control s Treatment S9 Plate counts DMS O 2 -Ni trofluorene DMS O 2 -Aminoanthracene 100 1/pl 2 g/pl 100 1/pl + 1 g/pl + 30 27 31 138 184 180 37 40 35 523 542 550 Mean 29 167 37 538 s. E. 1.2 14 . 7 1.5 8.0 * : Thinning o f the background l awn NT : Not tes ted RTC Study No. : 52400 Page 28 ASSAY ( S . typhimuri um and E . col i ) TABLE 1 0 - Experiment I - Plate incorporation method - TAl 0 0 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol Strain : TAl 0 0 Dose - level [ g/pl ] Without met abol i c act ivation Plate count s Mean S . E . Titre : 2 7 7 With metabo l i c activat ion Plate counts Mean S . E . Unt reated 0 . 00 156 313 625 1250 2500 5000 140 119 137 112 135 111 111 101 108 104 101 98 90 101 110 104 99 96 87 86 78 83 61 54 132 6 . 6 119 7 . 8 107 3 . 0 101 1 . 7 100 5 . 8 100 2 . 3 84 2 . 8 66 8 . 7 14 0 126 114 130 125 117 126 110 132 130 112 102 14 1 127 119 117 102 121 111 119 124 78 96 88 127 7 . 5 124 3 . 8 123 6 . 6 115 8 . 2 129 6 . 4 113 5 . 8 118 3 . 8 87 5 . 2 Regression analysis : Points S 9 Intercept 1-3 1 -4 1-5 1-6 1-7 1-3 + 1-4 + 1-5 + 1-6 + 1-7 + 10 . 860 10 . 686 10 . 520 10 . 515 10 . 477 11 . 184 10 . 976 11 . 103 11 . 042 11 . 180 S l ope -0 . 0028 -0 . 0013 -0 . 0006 -0 . 0006 -0 . 0005 -0 . 0014 0 . 0003 -0 . 0003 -0 . 0001 -0 . 0003 Corr . coe f f . -0 . 73205 -0 . 61350 -0 . 52691 -0 . 78101 -0 . 87495 - 0 . 38812 0 . 15202 - 0 . 24294 -0 . 19440 -0 . 74201 t 2 . 8430 2 . 4567 2 .2353 5 . 0024 7 . 8762 1 . 1142 0 . 4864 0 . 9030 0 . 7927 4 . 8246 P -value 0 . 02494 0 . 03387 0 . 04358 0 . 00013 0 . 00000 0 . 30197 0 . 63718 0 . 38296 0 . 43953 0 . 00012 Positive and negat ive controls Treatment Unt r e a t e d Sodium Az ide DMSO 2 -Aminoanthracene 1 g/pl 100 1/pl 1 g/pl S 9 Plate counts 14 0 119 137 681 796 722 + 118 119 124 + 1011 1449 1584 Mean S . E . 132 733 12 0 1348 6.6 33 . 7 1. 9 172 . 9 RTC Study No. : 52400 Page 29 7850 : BACTERIAL MUTATION ASSAY { S . typhimuri um and E . col i ) TABLE 1 1 - Experiment I I - Pre - incubat ion method - TA1 5 3 5 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S trai n : TA1 5 3 5 Dos e - l eve l [ g/p l ] Without metabo l i c act ivation Plate counts Mean s . E . Titre : 2 3 4 Unt reated 0 . 00 6 . 25 12 . 5 25 . 0 50 . 0 100 200 16 17 14 16 0 . 9 18 17 13 16 1 . 5 17 14 17 16 1 . 0 17 17 18 17 0 . 3 16 23 19 19 2 . 0 16 13 18 16 1 . 5 8 13 8 10 1 . 7 11 * 11 * 19 * 14 2 . 7 Regres s ion analys i s : Points S 9 Intercept 1 -3 1 -4 1 -5 1 -6 1 -7 3 . 964 3 . 948 4 . 096 4 .241 4 . 083 S l ope 0 . 0138 0 . 0169 0 . 0000 -0 . 0097 -0 . 0034 Corr . coeff . 0 . 33682 0 . 55260 0 . 00140 -0 . 68671 -0 .46161 t 0 . 9464 2 . 0967 0 . 0051 3 . 7787 2 . 2682 P -value 0 . 37545 0 . 06242 0 . 99604 0 . 00165 0 . 03516 Pos itive and negative control s Treatment S9 Plate count s Unt r e a t e d Sodium Az ide 1 g/pl 16 17 14 397 571 564 Mean 16 511 s. E. 0.9 56 . 9 * : Thinning o f the background l awn RTC Study No. : 52400 Page JO 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 1 2 - Experiment I I - Pre - incubation method - TA1 5 3 5 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : TA1 5 3 5 Dose - leve l [g/pl ] Titre : 2 34 With met abo l i c activat ion Plate counts Mean s . E . Unt r e a t e d 0 . 00 78 . 1 156 313 625 12 5 0 2500 5000 16 12 17 15 1 . 5 15 19 20 18 1.5 19 17 11 16 2 . 4 19 19 25 21 2.0 15 16 14 15 0 . 6 15 16 21 17 1 . 9 15 * 12 * 17 * 15 1 . 5 14 * 13 * 20 * 16 2 . 2 9 * 14 * 19 * 14 2 . 9 Regression analys i s : Point s S 9 Intercept 1-3 + 1-4 + 1-5 + 1-6 + 1-7 + 1-8 + 4 . 078 4 . 255 4 . 191 4 . 215 4 . 162 4 . 147 S l ope 0 . 0022 -0 . 0007 -0 . 0002 -0 . 0003 -0 . 0001 -0 . 0001 Corr . coeff . 0 . 31812 -0 . 20852 -0 . 09241 -0 . 31569 -0 .26043 -0 . 35396 t 0 . 8878 0 . 6742 0 . 3346 1 . 3308 1 . 1757 1 . 7751 P -value 0 . 4 0413 0 . 51545 0 . 74326 0 . 20190 0 . 25422 0 . 08972 Pos itive and negative controls Treatment S9 DMSO 2 -Aminoanthrac ene so 1/pl + 1 g/pl + Plate count s 16 15 17 99 103 82 Mean 16 95 s. E. 0.6 6.4 * : Thinning o f the background l awn RTC Study No. : 52400 Page 31 7850 : BACTERIAL MUTATION AS SAY ( S . typhimuri um and E . col i ) TABLE 1 3 - Experiment I I - Pre - incubat ion method - TA1 5 3 7 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : TA1 5 3 7 Dose - level [ g/p l ] Without metabo l i c act ivat ion Plate counts Mean s . E . Unt reated 0 . 00 3 . 13 6 . 25 12 . 5 25 . 0 50 . 0 100 19 22 17 19 1 . 5 16 12 19 16 2 . 0 14 19 19 17 1 . 7 14 16 15 15 0 . 6 21 15 17 18 1 . 8 15 19 14 16 1 . 5 21 13 * 15 16 * 18 14 * 18 14 1.7 0.9 Titre : 3 1 8 Regres sion analysi s : Points S 9 Intercept 1 -3 1 -4 1-5 1-6 1 -7 4 . 023 3 . 962 4 . 019 3 . 997 4 . 070 S l ope -0 . 0111 0 . 0143 0 . 0012 0 . 0041 -0 . 0017 Corr . coeff . -0 . 09346 0 . 2 1094 0 . 03439 0 .22852 -0 . 17753 t 0 . 2484 0 . 6824 0 . 1241 0 . 9389 0 . 7863 P -value 0 . 81098 0 . 51048 0 . 90316 0 . 36173 0 . 44136 Posi t ive and negative controls Treatment S9 Plate count s DMSO 9 -Aminoacridine 5 0 1/pl 50 g/pl 21 19 15 140 153 181 Mean 18 158 s. E. 1.8 12 . 1 * : Thinning o f the background l awn RTC Study No. : 52400 Page 32 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 1 4 - Experiment I I - Pre - incubat ion method - TA1 5 3 7 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : TA1 5 3 7 Dos e - level [g/pl ] Titre : 3 18 With metabolic act ivation Plate counts Mean s . E . Unt r e a t e d o . oo 78 . 1 156 313 625 1250 2500 5000 19 20 23 21 1.2 22 17 19 19 1 . 5 18 18 26 21 2 . 7 16 19 16 17 1 . 0 17 19 19 18 0 . 7 16 11 13 13 1 . 5 7* 6* 4* 6 0.9 10 * 3 * 5 * 6 2.1 5* 4* 8* 6 1.2 Regression analys is : Points S 9 Intercept 1-3 + 1-4 + 1-5 + 1-6 + 1-7 + 1-8 + 4 . 482 4 . 413 4 . 482 4 . 563 4 .330 4 . 068 S l ope -0 . 0017 -0 . 0006 - 0 . 0012 -0 . 0017 -0 . 0009 -0 . 0005 Corr . coeff . -0 . 33243 -0 . 23988 -0 . 68029 -0 . 91939 -0 . 84629 -0 . 75229 t 0 . 9326 0 . 7814 3 . 3465 9 . 3491 6 . 9246 5 . 3557 P -value 0 . 38209 0 .45268 0 . 00526 0 . 00000 0 . 00000 0 . 00002 Pos it ive and negat ive control s Treatment S9 DMSO 2 -Aminoanthracene 50 1/pl + 1 g/pl + Plate counts 20 22 23 74 98 95 Mean 22 89 s. E. 0.9 7.5 * : Thinning of the background l awn RTC Study No. : 52400 Page 33 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i } TABLE 1 5 - Experiment I I - Pre - incubat ion method - WP2 uvrA STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t ra in : WP2 uvrA Dose - leve l [ g/p l ] Without metabol i c activation Plate counts Mean s . E . Titre : 288 Unt r e a t e d 0 . 00 1 . 56 3 . 13 6 . 25 12 . S 25 . 0 50 . 0 30 18 25 20 26 25 26 18 27 27 33 32 19 30 34 22 24 30 25 26 29 33 24 32 24 3 . 5 24 1 . 9 24 2 . 8 31 1. 9 28 4.5 25 2 .4 27 1 . 2 30 2 . 8 Regres s ion analys i s : Points S9 Intercept 1 -3 1 1 - 4 5 1-6 1 -7 4 . 741 4 . 914 5 . 056 5 . 071 5 . 055 S l ope 0 . 2160 0 . 0732 0 . 0086 0 . 0044 0 . 0070 Corr . coeff . 0 . 60415 0 . 33244 0 . 07946 0 . 08531 0 .26029 t 2 . 0059 1. 1147 0 . 2874 0 . 3425 1 . 1751 P - va lue 0 . 08488 0 . 29107 0 . 77834 0 . 73645 0 . 25447 Pos itive and negative controls Treatment S9 Plate count s Unt reated MMS 500 g/pl 30 18 25 156 155 165 Mean 24 159 s. E. 3.5 3.2 RTC Study No. : 52400 Page 34 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 1 6 - Experiment I I - Pre - incubation method - WP2 uvrA STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : WP2 uvrA Dose - level [ g/p l ] Titre : 288 Wi th met abo l i c act ivation Pl ate count s Mean s . E . Un t r e a t e d 0 . 00 156 313 625 12 5 0 2500 5000 42 36 33 37 2 . 6 34 31 29 31 1.5 33 22 31 29 3.4 34 30 28 31 1. 8 29 24 22 25 2 . 1 31 * 39 * 33 * 34 2 . 4 22 * 30 * 33 * 28 3 . 3 20 * 28 * 27 * 25 2 . 5 Regres sion analys i s : Points S9 Intercept -1 - 3 + 1-4 + 1-5 + 1-6 + 1 7 + 5 . 518 5 . 599 5 . 366 5 . 447 5 . 4 92 S l ope -0 . 0002 -0 . 0009 0 . 0002 0 . 0000 -0 . 0001 Corr . coeff . -0 . 07463 -0 . 51241 0 . 2 1433 -0 . 03081 -0 . 32123 t 0 . 1980 1 . 8869 0 . 7912 0 . 1233 1 . 4786 P - va l ue 0 . 84867 0 . 08851 0 . 44304 0 . 90342 0 . 15564 Pos itive and negative controls Treatment S9 Plate count s DMSO 2 -Aminoanthrac ene 5 0 1/pl + 2 0 g/pl + 34 26 36 157 159 160 Mean 32 159 S. E. 3.1 0.9 * : Thinning o f the background l awn RTC Study No.: 52400 Page 35 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 1 7 - Experiment I I - Pre - incubat ion method - TA9 8 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol Strain : TA9 8 Dose - leve l [g/pl ] Without metabo l i c act ivation Plate counts Mean s . E . Titre : 3 1 0 With metabolic activat ion Plate counts Mean s . E . Unt r e a t e d o . oo 78 . 1 156 313 625 12 5 0 2500 5000 37 35 38 37 0 . 9 33 39 38 37 1 . 9 47 41 44 44 1. 7 47 SS 49 so 2 .4 38 41 46 42 2 . 3 37 38 44 40 2 . 2 39 34 35 36 1 . 5 25 * 30 * 22 * 26 2 . 3 NT NT NT 40 41 32 38 2 . 8 49 53 so 51 1.2 NT NT NT 51 48 so so 0 . 9 51 54 53 53 0 . 9 52 SS 47 51 2 . 3 43 * 46 * 42 * 44 1 . 2 43 * 40 * 46 * 43 1 . 7 26 * 34 * 28 * 29 2 . 4 Regress ion analysis : Points S 9 Intercept 1 -3 1 1 -- 4 5 1 1 - 6 7 1 1 - - 3 4 + + 1-5 + 1-6 + 1-7 + 6 . 071 6 . 412 6 . 548 6 . 592 6 . 646 7 . 070 7 . 108 7 . 226 7 . 178 7 . 225 S l ope 0 . 0067 0 . 0010 -0 . 0002 -0 . 0004 -0 . 0006 0 . 0004 0 .0001 -0 . 0004 -0 . 0003 -0 . 0003 Corr . coeff . 0 . 89046 0 . 27855 -0 . 09987 -0 . 42662 -0 .78926 0 .43290 0 . 19385 -0 . 66541 -0 . 75306 -0 . 92802 t 5 . 1772 0 . 9172 0 . 3619 1 . 8868 5 . 6025 1 . 2706 0 . 6249 3 . 2 14 0 4 . 5782 10 . 8590 P -value 0 . 00128 0 .38065 0 .72323 0 . 07747 0 . 00002 0 . 24448 0 . 54606 0 . 00678 0 .00031 0 . 00000 Positive and negat ive controls Treatment S9 Plate count s DMSO 2 -Nitrofluorene DMSO 2 -Aminoanthracene so 1/pl 2 g/pl so 1/pl + 2 g/pl + 38 35 30 145 141 134 44 41 36 422 439 450 Mean 34 14 0 40 437 s. E. 2.3 3.2 2.3 8.1 * : Thinning of the background l awn NT : Not t e s ted RTC Study No. : 52400 Page 36 7850 : BACTERIAL MUTATION AS SAY { S . typhimuri um and E . col i ) TABLE 1 8 - Experiment I I - Pre - incubat i on method - TAl 0 0 STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : TAl 0 0 Dose - level [g/pl ] Without metabo l i c act ivation Plate count s Mean s . E . Titre : 2 6 8 With metabo l i c activation Plate counts Mean s . E . Unt r e a t e d 0 . 00 78 . 1 156 3 13 625 1250 2500 5000 167 161 130 153 11 . 5 160 163 142 155 6 . 6 132 145 133 137 4 . 2 13 0 12 8 123 127 2 . 1 126 134 123 128 3 . 3 128 *117 * 99 * 115 8 . 5 109 *103 *105 * 106 1 . 8 68 * 84 * 96 * 83 8 . 1 NT NT NT 140 161 148 150 6 . 1 155 156 148 153 2 . 5 NT NT NT 143 133 120 132 6 . 7 131 111 121 121 5 . 8 124 114 117 118 3 . 0 133 *101 *107 * 114 9 . 8 98 * 94 *105 * 99 3 . 2 92 * 97 *102 * 97 2 . 9 Regres s ion analys i s : Points S9 Intercept 1-3 1 -4 1-5 1 -6 1 -7 1-3 + 1 1 -- 4 5 + + 1-6 + 1-7 + 12 . 388 12 . 135 12 . 019 11 . 856 11 . 764 12 . 301 12 . 037 11 . 796 11 . 661 11 . 433 S l ope -0 . 0075 -0 . 0034 -0 . 0023 -0 . 0014 -0 . 0011 -0 . 0044 -0 . 0022 -0 . 0011 -0 . 0008 -0 . 0004 Corr . coef f . -0 . 86130 -0 . 72247 -0 . 76000 -0 . 80731 -0 . 88582 -0 . 85246 -0 . 77707 -0 . 67730 -0 . 77706 -0 . 74335 t 4 .4849 3 . 3 044 4 . 2162 5 . 4721 8 . 3211 4 . 3 142 3 . 9041 3 . 3193 4 . 9382 4 . 8441 P -value 0 . 00285 0 . 00795 0 . 00101 0 . 00005 0 . 00000 0 . 00351 0 . 00294 0 . 00554 0 . 00015 0 . 00011 Pos itive and negative control s Treatment S9 Plate count s Unt r e a t e d Sodium Az ide DMSO 2 -Aminoanthracene 1 g/pl so 1/pl + 2 g/pl + 167 161 130 700 733 696 114 117 126 775 896 803 Mean 153 710 119 825 s. E. 11 . 5 11. 7 3 .6 36 . 6 * : Thinning of the background l awnu NT : Not te sted RTC Study No. : 52400 Page 3 7 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 1 9 - Experiment I I I - Plate incorpora t i on method - WP2 uvrA STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol Strain : WP2 uvrA Dose - l eve l [g/pl ] Without metabo l i c act ivation Plate counts Mean S . E . Titre : 2 8 1 Unt reated 0 . 00 50 . 0 100 200 400 800 25 19 19 21 2 . 0 24 24 21 23 1 . 0 22 22 20 21 0 . 7 30 25 24 26 1. 9 24 25 22 24 0 . 9 24 24 20 23 1 . 3 28 * 21 * 27 * 25 2 . 2 Regres s ion analys i s : Po int s S 9 Intercept 1 -3 1 -4 1-5 1-6 4 . 680 4 . 776 4 . 838 4 . 807 S l ope 0 . 0033 0 . 0008 0 . 0000 0 . 0002 Corr . coeff . 0 .48999 0 .25309 -0 . 02761 0 . 22713 t 1 . 4872 0 . 8273 0 . 0996 0 . 9329 P -value 0 . 18057 0 . 42738 0 . 92220 0 . 36474 Pos it ive and negative control s Treatment S9 Plate counts Unt reated MMS 5 0 0 g/pl 25 19 19 155 152 160 Mean 21 156 S. E. 2.0 2.3 * : Thinning o f the background lawn RTC Study No. : 52400 Page 38 7850 : BACTERIAL MUTATION ASSAY ( S . typhimuri um and E . col i ) TABLE 2 0 - Experiment I I I - Pre - incubat i on method - WP2 uvrA STUDY NO . : 5 2 4 0 0 SOLVENT : Ethanol S t rain : WP2 uvrA Dose - l evel [g/pl ] Without metabo l i c act ivat ion Plate count s Mean s . E . Unt r e a t e d 0 . 00 so . o 100 200 400 800 28 27 23 26 20 37 23 30 27 18 28 25 17 27 22 26 1.5 28 s.o 27 2 . 0 24 3 . 0 22 2 . 9 20 17 30 22 3 . 9 28 * 27 * 25 * 27 0 . 9 Titre : 2 8 1 Regre s s ion analys i s : Points S9 Intercept 1 -3 1 -4 1 -5 1 -6 5 . 260 5 . 228 5 . 118 4 . 958 S l ope -0 . 0037 -0 . 0029 -0. 0013 0 . 0000 Corr . coeff . -0 . 2 9637 -0 .41136 -0. 34695 -0 . 00095 t 0 . 8210 1 . 4272 1. 3338 0 . 0038 P -value 0 . 43871 0 . 18400 0 . 20517 0 . 99701 Pos itive and negative controls Treatment S9 Unt r e a t e d MMS s o 1/pl 500 g/pl Plate counts 28 27 23 126 137 144 Mean S . E . 26 1.5 136 5 . 2 * : Thinning of the background l awn RTC Study No. : 52400 Page 39 7850: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) APPENDIX 1 - Historical control data without metabolic activation STUDY NO. : 52400 Untreated TA1535 Mean value SD n TA1537 Mean value SD n TA98 Mean value SD n TAlOO Mean value SD n WP2uvrA Mean value SD n Plate incorporation 18 2.6 1 29 17 2. 1 128 32 3 .3 131 1 43 16.0 13 1 28 3.3 78 Untreated Pre-incubation 18 2.5 131 17 1.8 132 32 2.9 1 26 141 16.1 130 28 3.2 73 Positive control Plate incorporation 580 80.0 1 29 1 69 37.3 128 1 87 26.7 131 818 122. 1 131 1 84 1 8.7 78 Positive control Pre-incubation 579 79.3 131 1 62 47.6 132 181 26. 1 1 26 818 125.3 130 219 47.7 73 SD standard deviation n number of experiments RTC Study No. : 52400 Page 40 7850: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) APPENDIX 2 - Historical control data with metabolic activation STUDY NO. : 52400 TA1535 Mean value so n TA1537 Mean value so n TA98 Mean value so n TAl00 Mean value so n WP2uvrA Mean value so n Untreated Plate incorporation 16 1.8 1 29 21 2.6 128 39 3.7 13 1 1 53 1 6. 1 132 35 3.4 78 Untreated Pre-incubation 16 1 .9 128 21 2.2 133 40 3.0 118 1 50 15.1 130 34 3.3 71 Positive control Plate incorporation 141 30.3 1 29 120 2 1 .4 1 28 625 1 47 . 8 131 1278 198.8 132 203 37.4 78 SO standard deviation n number of experiments Positive control Pre-incubation 95 1 0.4 128 97 12.4 133 67 1 198.8 118 1 150 198.0 130 192 38.2 71 RTC Study No. : 52400 Page 41 7850: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) ADDENDUM I - Certificate of analysis STUDY NO.: 52400 RTC Study No. : 52400 Page 42 (I) SOLVAY SOLEXIS C.D. C. 03 1 6 Commessa N751 Centro Ricerche e Svilup Viale Lombardia, 20 20021 Bollate (Ml) Richiesta analisi Numero: Risposta Numero: Richiedente Pag. Data 23/1 1/2004 Data Destinatario Prodotto, suoi riferimenti e precauzioni antinfortunistiche ---7850 Acido Batch: 3223 ON Tossico e corrosive: utilizzare guanti e occhiali, lavorare sotto cappa I Lavoro richiesto Caratterizzazione NMR: analisi terminali, peso molecolare Risulta ti Si allega spettro 1 9F-NMR con caratterizzazione del prodotto Si nota la presenza di piccolissime quantita di estere Distribuzione Riferim 1 1 542 RTC Study No. : 52400 Page 43 :::ti h FLX;:5!0_322'3ml.. PfP8 exp2 112pu l SAMPL3 DEC. I VT dllt.cr 11oY lO 20H dftq 299.,, ant.vent Acetont, dn Kl ....IIYP1D8 .. dpvz JO U.U50.. dot 0 _32230N_o01 . !:Ld Gl .... - ACQUISl'tICl,I C fzq ttJ . 11i drat 200 tn Flt P'Rtx:USlNli ., Vo o . soc lb : ; oo . t-..J 1!,110 lfid -1 .,. C ,.su a . o wt:filt 4 1.400 pi:OC lp .. .. ,. C 1G 111012 tpwr ,.,pv dt nt ot .alaet .4 , .'5 2 . (100 .2400,0 1000 1000 _,rr xp till> wt.t ruet p.rooplot gdn tl ln AOt UHd !UGO . DISPt.AY P -451 4 & . , wp 3'502 . 5 . ,,0 250 .... aa.01 ia 483. !I? rfl 229'65. rfp -Jts1a.a ' th ,.., 140..0S .i IS=4 84 FLK7850_32230N_001 , jpeg .MOL ,llEIGliT 551 EQUIV. lff:IGHT = 5 5 1 Cll. O 1. C3 C2 9 . l "/, C3 -ocr20- -ocr <CF3l o- --- -ocF3 -OCFCFJCOOH -OCF2CL -<iCF ( CF3) CF2CL = -OCF2CF ( CFJ ) CL -OCF2COOli -= J.J o. 8 1 .l 15,B 29.7 49.2 l.I._... Page FLll'l 8 5 0_322 30N__ PFPE Pulse Sequence : s2pul I I I I I i I I Ii II) I I III I II I i II I I I IJ "1::t -30 -40 -50 -60 -70 -80 - 90 - 1 0 0 - 1 1 0 -120 - 1 3 0 -1 4 0 - 1 5 0 ppm L...-r-- 0 . 32 -o . oo 1 . 14 -0 ..1 9 120 . 00 o. 51 1 8 . so 7850: BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) ADDENDUM II - Study Protocol STUDY NO. : 52400 RTC Study No. : 52400 Page 45 \IN - rjrri CI \INI \ Aq , \_ F\ I Li RESEARCH TOXICOLOGY CENTRE - ROMA Version 05/2 7850 BACTERIAL MUTATION ASSAY (S. typhimurium and E. coli) Final Protocol prepared for SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (MI) Italy by RESEARCH TOXICOLOGY CENTRE S.p.A. Via Tito Speri, 12 00040 Pomezia (Roma) Italy RTC Enquiry Number: 52400 Cenumensiall &Moe RTCS o.A Vie Trto Saari. 12 0 0040 Pomezia (Roma) - ITALY Tel.: 39 08 91095 1 Fax. 39 08.910.5737 e-mail ippon sirevcrt RTC Study No.: 52400 - 1 of 13 - Mud Office red Administration RTC So-AVia Teo Speri. 12 00040 Pomezia (Retrial - ITALY Tel.: 3908.91095.1 Fax: + 3906.912.2233 P.O. Box 15301-00143 - Roma For LaurentIno January 2006 RTC S.p.A. Capital." eoc+ale Euro 5.164.000 C.C.I.A.A. n' 375376 Reg. Soc. Trib. di Roma a' 2828/72 Cod. Fele.. 00653120584 Partite IVA: 00920611001 Page 46 Version 05/2 BACTERIAL MUTATION ASSAY (S. typhimurium and E. col,) MANAGEMENT OF STUDY Scientific Director Head of Genetic and Cellular Toxicology Study Director Sponsor SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 2002 1 Bollate (MQ Italy Monitor QUALITY ASSURANCE Quality Assurance Manager LOCATION OF STUDY The study will be performed at Research Toxicology Centre S.p.A. Via Tito Sperl, 1 2 00040 Pomezia (Roma) Italy The laboratory facilities, archives and administration are located at this site. TIME SCHEDULE OF STUDY The Study will be conducted with a time schedule agreed between the Sponsor and RTC. TEST ITEM IDENTITY The test item will be 7850 RTC Enquiry Number: 52400 RTC Study No. : 52400 - 2 of 1 3 January 2006 Page 47 Version 05/2 BACTERIAL MUTATION ASSAY (S. typhimurium and E. coll) 1. INTRODUCTION 1.1 Objective To assay the test item for the ability to induce gene mutations in Salmonella_typhimurium and Escherichia coli, 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 2004/1 0/EC of the European Parliament and of the Council of 1 1 February 2004; - ENV/MC/CHEM(98)1 7 "OECD principles on Good Laboratory Practice - as revised in 1 997"; - Decreto Legislativo no. 120 of27 January 1992 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. 47 1 (Adopted July 1 997). - 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. coli tester strains ofthe 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: 52400 - 3 of 1 3 January 2006 RTC Study No. : 52400 Page 48 Version 05/2 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 solutions of the test item will be prepared for each day' s wotk; solutions will be prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test item. Unless specified by the Sponsor, concentrations of solutions will be expressed in terms of material as received, and not of active constituents. Preferred solvents will be sterile distilled water, culture medium, DMSO, ethanol, acetone. Other solvents may be used as necessary. 2.8 No assay of 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 1 5 35, TA 1 537, TA 1 53 8 , TA 98, TA 100, TA 97 and TA 1 02 and some other related strains) were obtained from University of California. Stocks of E. coli tester strains (WP2, WP2 uvrA and WP2 uvrA pKM l 0 l ) 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 1 09 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.M I O l 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: 52400 - 4 of 13 January 2006 RTC Study No. : 52400 Page 49 Version 05/2 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. 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 Difeo Bacto-agar ( 1 5g) 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% Difeo Bactci-agar in Vogel Bonner Medium E, with 2% Glucose, and poured into plastic Petri dishes. Top Agar: "Top Agar" (overlay agar) will be prepared as 0.6% Difeo Bacto-agar + 0.5% NaCl 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 1 00 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 1 254 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 ( l 00 mM) G-6-P ( 1 00 mM) KCl (330 mM) MgC12 ( 1 00 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 1 0 .0 ml RTC Enquiry Number: 5 2400 - 5 of 1 3 January 2006 R TC Study No. : 52400 Page 50 Version 05/2 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 wi11 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 (1 0%) 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 TA1 535, TA1537, TA98, TAl 00 and E. coli WP2 uvrA will be used in this study. RTC Enquiry Number: 52400 RTC Study No. : 52400 - 6 of 13 January 2006 Page 51 Version 05/2 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 Preseace ofS9 TA1 535 sodium azide 1 g/plate 2-aminoanthracene 1 g/plate TAl 00 sodium azide I g/plate 2-aminoanthracene 1 g/plate (2 g/plate) TA 1 5 37 9-amino-acridine SO 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 I0 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 oftwo. Where it seems advisable, further test points or controls may be included in experiments. In addition, plates will be prepared to check the sterility ofthe test item solutions and the S9 mix, and dilutions of the bacterial cultures will be plated on nutrient agar plates to establish the number ofbacteria in the cultures. 6. ASSAY PROCEDURE 6.1 Plate-incorporation The components ofthe 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: 52400 RTC Study No.: 52400 - 7 of 13 January 2006 Page 52 Version 05/2 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 (iv) Bacterial suspension 2 ml 0.1 ml 0.5 ml 0.1 ml 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 (Pb 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: 52400 RTC Study No. : 52400 - 8 of 13 - January 2006 Page 53 Version 05/2 7.2 Evaluation of data For the test item to be consideredmutagenic, two-fold(or more) increases in mean revertant numbers must be observedat 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 ofmutant colonies with increasing dose-levels. Evaluation of Ames test data basedon a 'doubling rate' has been shown to be as effective as statistical techniques in allowing the correct interpretation of test results (Chu et al. 1 981). 7.3 Reporting procedure A Draft Report will be supplied, and a Final Report issued subsequently to include any agreed changes or amendments. If any corrections or additions are requiredto 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 addedto or corrected, andthe reasons for the changes, andwill be signedanddatedby the person responsible. 7 .4 Final report The following information anddata will be includedin the final report: - name andaddress of the facility performing the study andthe dates on which the study was initiatedandcompleted; - objective andprocedures statedin the approvedprotocol, including approvedchanges to the original protocol; - the test article, identifiedby name, chemical name or chemical number; - methodused; - any unforeseen circumstances that may have affectedthe quality or integrity of the study; - data generatedwhile conducting the study; - statistical methods employedfor analysing the data; - a summary of the data, an analysis of the data anda statement of the conclusions drawn from the analysis; historical untreatedand positive control data; the name andsignature 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 boundanda PDF version will be supplied. RTC Enquiry Number: 52400 - 9 of 13 - January 2006 RTC Study No. : 52400 Page 54 Version 05/2 7.5 Records kept Full records will be maintainedof all aspects of study conduct, along with the results of all measurements andobservations. Prior to final archiving of the study data a full list will be preparedof all records associatedwith the study. 7.6 Archiving All raw data, records and documentation arising from this study anda copy of the final report consigned, generated during the course of this study will be retained at RTC. Archiving will be providedfor a periodof 3 years after which the Sponsor will be contacted for instructions regarding despatch or disposal of the material. As a further option, archiving space can be rentedfor an additional time. The signedFinal Protocol and the top copy of the Final R eport will be despatchedto andarchivedby the Sponsor. 8. STUDY CONDUCT 8. l Language English language andItalian language versions of the study protocol, Standard Operating Procedures andother study documents may be usedinterchangeably. Similarly, English and Italian renderings of chemical names, including that of the test material will be consideredto 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 describedfor the selection of a solvent, selection of dose levels, interpretation of the outcome of the study or other aspects of the study conduct, he will recordcarefully the decision he has reachedandthe reasoning which ledto it. 8.3 Quality assurance The study is subjectedto the procedure for quality assurance as definedby the relevant GLP regulations. Specifically: - the protocol is inspectedfor compliance; - procedures of the laboratories concernedwill be inspectedat intervals adequate to assure the integrity of the study; - the final report is reviewedto ensure that it accurately describes the methods andrelevant StandardOperating Procedures andthat the results are in agreement with the raw data; - periodic reports on these activities are made to management andthe Study Director. All raw data pertaining to the study will be available for inspection by the study monitor (for scientific monitoring) or the Quality Assurance Unit of the Sponsor (compliance monitoring). RTC Enquiry Number: 52400 - 1 0 of 1 3 - January 2006 RTC Study No. : 52400 Page 55 Version 05/2 9. DEPARTURES FROM REGULATORY REQUIREMENTS Items which are the responsibility of the Sponsor are indicated in sections 2. 1 , 2.4, 2.5, 2.7, 2.8 and 3.3 of this protocol. Since full compliance with regulatory requirements may depend on the performance of these items, the Sponsor should ensure that appropriate actions are initiated or undertaken. RTC Enquiry Number: 52400 RTC Study No. : 52400 - 1 1 of 13 - January 2006 Page 56 Version 05/2 10. REFERENCES Arnes,BN, J. McCann and E. Yamasaki ( 1 975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Research 3 1 , 347-364. Chu K.C. et al. (1981) Evaluating statistical analyses and reproducibility ofmicrobial mutagenicity assays. Mutation Research 85, 1 19-1 32. Claxton L.D. et al (1 987) Guide for the Salmonella typhimuriumlmammalian microsome tests for bacterial mutagenicity. Mutation Research 1 89, 83-9 1 . Gatehouse D . et al. (1994) Recommendations for the performance ofbacterial mutation assays. Mutation Research 3 1 2, 2 1 7-233. Green M.H.L. and W.J. Muriel (1976) Mutagen testing using TRP+ reversion in Escherichia coli. Mutation Research 38, 3-32. Maron D.M. and B.N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research 1 1 3, 1 73-215. 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, 1 983. Venitt S . , C . Croften-Sleigh and R. Forster ( 1 984) Bacterial mutation assays using reverse mutation in: Mutagenicity Testing - a practical approach S. Venitt and J.M. Parry (eds.) IRL Press, Oxford, 1 984. RTC Enquiry Number: 52400 RTC Study No.: 52400 - 12 of 13 January 2006 Page 57 STUDY TI1LE TEST FACILITY RTC STUDY NO. TEST ITEM APPROVED BY Version 05/2 APPROVAL PAGE BACTERIAL MUTATION ASSAY (S. typhimuriurn and E. coli) RESEARCH TOXICOLOGY CENTRE S.p.A. Via Tito Speri, 12 00040 Pomezia (RM) Italy 52400 RELEASED BY Date SPONSOR AUTHORISED BY SPONSOR SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 2002 1 Bollate (Ml) Italy Name and Title * Please print or type your name and company status below your signature RTC Enquiry Number: 52400 RTCStudy No. : 52400 - 13 of 13 - January 2006 Page 58