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4 A, --44i5WL kiiiV- iO- N\NY N'Air" ..1tIV\P14/1\JkAk RESEARCH TOXICOLOGY CENTRE ROMA 7850 MUTATION IN L5178Y TKO"'' MOUSE LYMPHOMA CELLS (FLUCTUATION METHOD) FINAL REPORT RTC Study no.: 52410 Sponsor: SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (M1) Italy Total number of pages: 72 Commercial Office RTC S.p.A. -Via Tito Sped, 12 00040 Pomezia (Roma) - ITALY Tel.: +39.06.91095.1 Fax: +39.06.910.5737 e-mail: Il@rtc.it www.rtc rt Head Office and Administration RTC S.P.A. Via Tito Spec, 12 00040 Pomezia (Roma) - ITALY Tel.: + 39.06.91095.1 Fax: + 39.06.9122233 P.O. Rex 15301-00143 - Roma Ear Laurentino RTC S.p.A. Gapitale sociafe Euro 5.164.000 C.C.LA.A. 375375 Reg. Soc. Trib. di Roma n' 2828/72 Cod. Fisc, 00653120584 Partite iVA: 00929611001 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 Legislative 27 Gennaio 1992 n. 120, Adoption o/88/320/EEC and 90/18/EEC Directives on the inspection and verification ofgood laboratory practice (G. U. 18 Febbraio 1992 n. 40) and subsequent revisions. C. Directive 2004/10/EC of European Parliament and of the Council of 11 February 2004. On the harmonisation of laws, regulations and administrative provisions relating to the application of the principles ofgood laboratory practice and the verification of their applications for tests on chemical substances. D. ENV/MC/CHEM(98) 17 OECD principles on Good Laboratory Practice (as revised in 1997). Date: Date: RTC Study No.: 52410 Page2 QUALITY ASSURANCE STATEMENT (Relevant to the aspects of the study conducted by RTC) Study phases monitored by RTC's QAU according to current relevant Standard Operating Procedures PROTOCOL CHECK Quality Assurance Inspections (Day Month Year) Inspection 06.02.2006 Report to Study Director 07.02.2006 Report to Company Management 07.02.2006 PROCESS-BASED INSPECTIONS RELATED TO THIS TYPE OF STUDY Dose preparation Cell treatment Count and plating out Plate scoring 04.05.2006 15.03.2006 03.05.2006 04.05.2006 11.05.2006 17.03.2006 06.07.2006 04.05.2006 Other process-based inspections were carried out on routine activities not directly related to this type of study. The relevant documentation is kept on file although specific inspection dates are not reported here. Associated laboratories and support functions are subject to regular facility inspections. FINAL REPORT Review of this report by RTC's QAU found the reported methods and procedures to describe those used and the results to constitute an accurate representation of the recorded raw data. Review completed Date RTC Study No.: 52410 Page3 Contents Page 1. SUMMARY ................................................................................................................................5 2. INTRODUCTION.......................................................................................................................6 2.1 Purpose..............................................................................................................................6 2.2 Principles of the method ...................................................................................................6 2.3 Study organisation ............................................................................................................7 3. MATERIALS..............................................................................................................................8 3.1 Test item ...........................................................................................................................8 3.2 Control items.....................................................................................................................8 3.3 S9 Tissue homogenate ......................................................................................................9 3.4 L5178Y TK+/- mouse lymphoma cells ............................................................................9 3.5 Culture media..................................................................................................................10 4. METHODS ...............................................................................................................................11 4.1 Preparation of test cell cultures.......................................................................................11 4.2 Cytotoxicity assay ...........................................................................................................11 4.3 Mutation assay ................................................................................................................12 4.3.1 Treatment of cell cultures ...............................................................................................12 4.3.2 Determination of survival ...............................................................................................12 4.3.3 Expression period............................................................................................................12 4.3.4 Plating for 5-trifluorothymidine resistance .....................................................................12 4.3.5 Plating for viability .........................................................................................................12 5. ANALYSIS OF RESULTS.......................................................................................................13 5.1 Survival or viability ........................................................................................................13 5.2 Total suspension growth and relative total growth .........................................................13 5.3 Growth factor over two days...........................................................................................14 5.4 Mutation frequency.........................................................................................................14 5.5 Statistical analysis...........................................................................................................14 5.5.1 Test for consistency between plates................................................................................14 5.5.2 Heterogeneity factors for replicate cultures ....................................................................15 5.5.3 Test for overall consistency ............................................................................................15 5.5.4 Updated heterogeneity factors ........................................................................................15 5.5.5 Comparison of each treatment with the control ..............................................................16 5.5.6 Test for linear trend.........................................................................................................16 5.6 Acceptance criteria..........................................................................................................16 5.7 Criteria for outcome of assay ..........................................................................................17 6. RESULTS .................................................................................................................................18 6.1 Solubility test ..................................................................................................................18 6.2 Cytotoxicity test ..............................................................................................................18 6.3 Mutation assays...............................................................................................................19 6.4 Osmolality and pH ..........................................................................................................20 7. CONCLUSIONS.......................................................................................................................21 8. REFERENCES..........................................................................................................................22 9. TABLES 1 TO 19 .....................................................................................................................23 10. APPENDIX I - Statistical analysis............................................................................................43 11. APPENDIX II - Historical data.................................................................................................50 12. APPENDIX III - Certificate of analysis....................................................................................51 13. APPENDIX IV - Study Protocol...............................................................................................54 RTC Study No.: 52410 Page 4 1. SUMMARY The test item 7850 was examined for mutagenic activity by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment (in the absence and presence of S9 metabolic activation) using a fluctuation method. Solutions of the test item were prepared in ethanol. A preliminary cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose-level of 5000 g/ml and at a wide range of lower dose-levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 g/ml. Following treatment in the absence of S9 metabolic activation, using a 3-hour treatment time, no cells survived at dose levels between 156 and 5000 g/ml. Slight toxicity was observed over the remaining dose levels reducing survival to 74% of the concurrent negative control at 78.1 g/ml. Using a long treatment time, no cell survived at concentrations between 313 and 5000 g/ml, while at the next lower doselevel (156 g/ml) survival was reduced to 44% of the negative control value. Following treatment in the presence of S9 metabolic activation, no cells survived at concentrations between 313 and 5000 g/ml. Dose-related toxicity was observed over the remaining dose levels, reducing survival to 8% of the negative control value at 156 g/ml. Two independent assays for mutation at the TK locus were performed using dose levels described in the following table: Assay No.: S9 1 - Treatment Time (hours) 3 Dose-level (g/ml) 125, 100, 80.0, 40.0, 20.0, 10.0 and 5.00 1 + 3 150, 100, 75.0, 37.5, 18.8 and 9.38, 2 - 24 160, 120, 80.0, 40.0, 20.0 and 10.0 2 + 3 120, 100, 83.3, 69.4 and 57.9 No mutant frequency above those seen in the concurrent vehicle control or historical background ranges was observed following treatment with the test item, in the absence or presence of S9 metabolism. Untreated, vehicle and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the untreated control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that 7850 does not induce mutation in mouse lymphoma L5178Y cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions. RTC Study No.: 52410 Page 5 2. INTRODUCTION 2.1 Purpose This report describes experiments performed to assess the mutagenic activity of the test item by assaying for the induction of 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment (in the absence and presence of S9 metabolic activation) using a fluctuation method. This method may detect gene mutation, clastogenic and aneugenic effects. The study was designed to comply with the experimental methods indicated in: EEC Council Directive 2000/32 Annex 4E. OECD Guideline for the testing of chemicals No. 476 (adopted July 1997). ICH, Topic S2A, Genotoxicity: Specific Aspects of Regulatory Tests, Step 4 (Document, July 1995). ICH, Topic S2B, A Standard Battery for Genotoxicity Testing of Pharmaceuticals, Step 4 (Document, July 1997). 2.2 Principles of the method The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single step forward mutation to the TK-/- genotype in which little or no TK activity remains. The cells used, L5178Y TK+/-, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been valuated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells. RTC Study No.: 52410 Page 6 2.3 Study organisation Sponsor SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 20021 Bollate (MI) Italy Test facility Genetic and Cellular Toxicology Department Research Toxicology Centre S.p.A. Via Tito Speri, 12 00040 Pomezia (Rome) Italy Principal dates Study protocol approved by Study Director: 18-Jan-2006 Experimental starting date: 06-Mar-2006 (Treatment - Toxicity test) Experimental completion date: 04-May-2006 (End of scoring - Main Assay 2) Personnel involved in the study Study Monitor: Study Director: Archiving The original data arising from this study 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, 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.: 52410 Page 7 3. MATERIALS 3.1 Test item Details of the test item received at RTC were as follows: Name Batch C.A.S. No. Received from Date received Amount received Description Expiry date Container Storage at RTC RTC reference number : 7850 : 3223 ON : 329238-24-6 : SOLVAY SOLEXIS : 20-Feb-2006 : 100 g : Colourless liquid : 31-Dec-2012 : Opaque plastic bottle : Room temperature : 10022 On 27-Feb-2006 a 12g sub-sample of the test item was transferred from the Formulation Unit to the Department of Genetic and Cellular Toxicology and stored under the same conditions. A certificate of analysis, supplied by the Sponsor, can be found in Appendix IV of this report. Solutions of 7850, as received, were prepared immediately before use in ethanol 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 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 solvent used in this study was ethanol obtained from 4D03334D). (batch nos.: 4G297304I and Fresh solutions of methylmethanesulphonate (MMS) (batch no.: 359316/153696 obtained from Fluka AG) were prepared in sterile distilled water and served as positive controls in the absence of S9 metabolism. Fresh solutions of benzo(a)pyrene (B(a)P) (batch no.: 18H3486 obtained from Sigma Chemical Co.) were prepared in dimethylsulfoxide (DMSO) and served as positive controls in the presence of S9 metabolism. Injectable grade distilled water (batch nos.: 03H2801 and 03H2802) was obtained from Bieffe, Trieste, Italy. DMSO (batch nos.: 1060564 41204083) was obtained from Fluka AG. Untreated cultures were included for each treatment series and acted as concurrent controls for the positive control treatments. RTC Study No.: 52410 Page 8 3.3 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 (M/g 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 TA100. Induced Salmonella typhimurium revertants: TA100 2-Aminoanthracene (1 g/plate) Benzo(a)pyrene (2.5 g/plate) 1557 197 514 27 The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 ml): S9 tissue fraction NADP (0.03 M) G-6-P (0.59 M) KCl (150 mM) Complete medium (5%) 0.408 ml 0.204 ml 0.204 ml 0.204 ml 8.98 ml 10.0 ml 3.4 L5178Y TK+/- mouse lymphoma cells L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination. Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Prior to use cells were cleansed of preexisting mutants. RTC Study No.: 52410 Page 9 3.5 Culture media The following culture media were used: Minimal medium A RPMI 1640 (1X) L-glutamine (200 mM) Sodium pyruvate (100 mM) Non-essential amino acids (100X) Streptomycin sulphate 50.000 IU/ml Penicillin G 50.000 units/ml F 68 Pluronic 516.1 ml 5.4 ml 6.0 ml 5.4 ml 1.1 ml 6.0 ml Minimal medium B RPMI 1640 (1X) L-glutamine (200 mM) Sodium pyruvate (100 mM) Non-essential amino acids (100X) Streptomycin sulphate 50.000 units/ml Penicillin G 50.000 units/ml 522.1 ml 5.4 ml 6.0 ml 5.4 ml 1.1 ml Complete medium (5%) Minimal medium A Horse serum (heat-inactivated) 950 ml 50 ml Complete medium (10%) Minimal medium A Horse serum (heat-inactivated) 900 ml 100 ml Complete medium A (20%) Minimal medium A Horse serum (heat-inactivated) 800 ml 200 ml Complete medium B (20%) Minimal medium B Horse serum (heat-inactivated) 800 ml 200 ml RTC Study No.: 52410 Page 10 4. METHODS 4.1 Preparation of test cell cultures A cell suspension (1 x 106 cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes. The treatment media were prepared as follows: Without S9 metabolism 3-hour treatment time Cell suspension (1 x 106 cells/ml in complete medium 5%) Complete medium (5%) Control or Test item solution Without S9 metabolism 24-hour treatment time 10.0 ml 9.8 ml 0.2 ml 20.0 ml Cell suspension (1 x 106 cells/ml in complete medium 10%) Complete medium (10%) Control or Test item solution With S9 metabolism 3-hour treatment time 3.0 ml 16.8 ml 0.2 ml 20.0 ml Cell suspension (1 x 106 cells/ml in complete medium 5%) S9 mix Control or Test item solution 10.0 ml 9.8 ml 0.2 ml 20.0 ml The cultures were incubated at 37C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS). 4.2 Cytotoxicity assay A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and in the presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20 ml RPMI minimal medium A. Cell concentrations were adjusted to 8 cells/ml using complete medium (20%) and, for each dose level, 0.2 ml was plated into 96 microtitre wells. The plates were incubated at 37C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8-9 days. Wells containing viable clones were identified by the eye using background illumination and then counted. RTC Study No.: 52410 Page 11 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 Mutation assay Treatment of cell cultures Experiments were performed including untreated, vehicle and positive controls, in the absence and presence of S9 metabolising system. Preparation of test cell cultures was performed as described in section 4.1. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a long treatment time (24 hours). After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2 x 105 cells/ml. The cultures were incubated at 37C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype. Determination of survival Following adjustment of the cell densities, samples of the cultures were diluted to 8 cell/ml using complete medium A (20%). A 0.2 ml aliquot of each diluted culture was placed into each well of two 96-well plates. The plates were incubated at 37C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. After incubation, wells containing viable clones were identified by the eye using background illumination and then counted. Expression period During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2 x 105 cells/ml. Plating for 5-trifluorothymidine resistance After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 g/ml) and an estimated 2 x 103 cells was plated in each well of four 96-well plates. Plates were incubated at 37C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as described in section 4.3.2 and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored. Plating for viability After dilution, in complete medium A (20%), an estimated 1.6 cells/well was plated in each well of two 96-well plates. These plates were incubated at 37C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted. RTC Study No.: 52410 Page 12 5. ANALYSIS OF RESULTS 5.1 Survival or viability The following formula was used to determine the plating efficiency (PE): PE = -ln (fyss/ns) where: ys = Number of empty wells (without clones) ns = Total number of wells fs = Number of cells plated per well (1.6 cells/well) Day 1 plating efficiencies in test and control cultures were compared to give the relative plating efficiency (RPE). Since reduction in post-treatment cell count was observed, relative survival calculation was corrected as follows: %RS = RPE x cell count factor where: cell count factor = treated post-treatment cell count control post-treatment cell count 5.2 Total suspension growth and relative total growth In order to aid toxicity data interpretation the relative total growth (RTG), expressed as a percentage of the concurrent negative control, was also calculated. This is a product of the relative suspension growth (RSG) and the Day 2 relative plating efficiency (RPE), expressed as a percentage of the concurrent negative control, for each culture, as follows: RTG = RSG x RPE (Day 2) Relative suspension growth (RSG) is given by: [TSG(test) / TSG (control)] x 100 where TSG (total suspension growth) is calculated as follows: TSG = [post-treatment cell count] [pre-treatment cell count] x [Day 1 cell count] [2 x 105*] x * Or appropriate cell concentration if lower [Day 2 cell count] [2 x 105*] RTC Study No.: 52410 Page 13 5.3 5.4 5.5 5.5.1 Growth factor over two days The suspension growth factor (SG) over two days is a parameter to evaluate the number of generations during the phenotypic expression time and is calculated as follows: SG = [Day 1 cell count] [2 x 105*] [Day 2 cell count] x [2 x 105*] * Or appropriate cell concentration if lower Mutation frequency Mutant frequencies (MF) for each treatment were calculated using Poisson statistics. The following formula was used: MF = [PE(mutant)/PE(viable)] x 106 Therefore: MF = { -ln (ym/nm) / -ln (ys/ns) } x 106 fm fs where: ym = Number of empty wells (mutant plates) nm = Total number of wells (mutant plates) fm = Number of cells plated per well (2x103 cell/well) ys = Number of empty wells (viability plates) ns = Total number of wells (viability plates) fs = Number of cells plated per well (1.6 cells/well) Statistical analysis Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990). Test for consistency between plates Results of individual plates within a replicate treatment were checked for consistency by calculation of the term 2 as follows: 2 = N(N[yi2/ni]-Y2) Y(N-Y) where: N = Total number of wells Y = Total number of empty wells ni = Total number of wells in the ith plate yi = Total number of empty wells in the ith plate 2 was compared with the critical values of the chi-squared distribution ( = 0.001) with M-1 degrees of freedom, where M is the number of plates used. RTC Study No.: 52410 Page 14 5.5.2 Heterogeneity factors for replicate cultures For negative control and test item treatments, the consistency between replicate cultures was evaluated by calculation of the heterogeneity factor (H) from a table constructed as follows: Culture A Culture B Sum Empty wells yA yB Y = yA + yB Total wells nA nB N = nA + nB Proportion pA = yA / nA pB = yB / nB P = Y / N 5.5.3 5.5.4 Where: d = pA - pB V = P(1 - P)(1/ nA + 1/ nB) and H = d2 V The heterogeneity factors (H) were calculated for survival (Hs), viability (Hv) and mutation (Hm). Values obtained should not exceed 10.8 times the current heterogeneity factor where 10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinite degrees of freedom. Test for overall consistency The overall consistency was evaluated by the calculation of the following ratio: H experiment / current heterogeneity factor where H experiment are the mean values for Hs, Hv and Hm. This ratio should not exceed the one-sided 1% critical values from the F-distribution (the number of degrees of freedom at the numerator is equal to the number of pairs of cultures, whereas the number of degrees of freedom at the denominator is infinite). Updated heterogeneity factors The estimated H experiment values were combined with the current heterogeneity factors to define the updated estimate factors as follows: Updated heterogeneity = 1/20 H experiment + 19/20 current heterogeneity RTC Study No.: 52410 Page 15 5.5.5 5.5.6 5.6 Comparison of each treatment with the control The control log mutant frequency (LMFc) was compared with the log mutant frequency from each treatment dose (LMFi) by calculation of: Di2 var(Di) where: Di = LMFi - LMFc var(Di) = Vi + Vc Vi and Vc are the variances of the treatments and control LMF. The heterogeneity factors were used to modify the estimate of variances. For each comparison of treatment with control, the ratio Di2/var(Di) was compared to the critical values for the one tailed Dunnetts test. Test for linear trend The evaluation of a linear trend in mutant frequency with treatment dose was performed using weighted regression. The weights were obtained from the variances of LMF (V) as follows: W= 1 _ V (MF)2 The slope b and its variance var(b) were calculated to form the test statistic b2/var(b) which was compared with tabulated critical values of 2 with 1 degree of freedom. Acceptance criteria The assay was considered valid if the following criteria were met: (i) The cloning efficiencies at Day 2 in the untreated control cultures in the absence of S9 metabolic activation fell within the range of 65-120%. (ii) The untreated control growth factor in the absence of S9 metabolic activation over 2 days fell within the range of 8 32. (iii) The mutant frequencies in the untreated control cultures fell within the range of 50-200 x 106 viable cells. (iv) The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). RTC Study No.: 52410 Page 16 5.7 Criteria for outcome of assay For a test item to be considered mutagenic in this assay, it is required that: (i) The mutant frequency at one or more doses is statistically significantly greater than that of the negative control. (ii) There is a significant dose-relationship as indicated by the linear trend analysis. Any increase in mutant frequency should lie outside the historical control range to have biological relevance. RTC Study No.: 52410 Page 17 6. RESULTS 6.1 Solubility test As indicated by the Sponsor, the test item was found to be miscible in ethanol at a concentration of 500 mg/ml. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity. A dilution series in ethanol was prepared and aliquots were added to RPMI complete medium (10%) in the ratio 1 : 100. The addition of a stock solution at 500 mg/ml generated precipitation, while an aliquot at 250 mg/ml generated a clear solution. On the basis of this result a concentration of 5000 g/ml was selected as the highest dose level to be used in the cytotoxicity test. During the toxicity test, upon addition of 7850 to the cultures a gel formation was observed at the dose levels of 5000 and 2500 g/ml, both in the absence and presence of S9 metabolism. At the end of the treatment incubation period, precipitation was observed at the dose levels of 5000, 2500, 1250 and 625 g/ml in the presence of S9. During the first experiment no precipitation was observed at the beginning of treatment, while precipitation was observed at the end of the treatment incubation period at concentration levels between 80.0 and 150 g/ml. During the second experiment precipitation was observed at the end of the treatment incubation period at the three highest dose levels, in the presence of S9 metabolic activation. 6.2 Cytotoxicity test Both in the absence and presence of S9 metabolism, the test item was assayed at a maximum concentration of 5000 g/ml and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 g/ml. The cell count factors, the raw plate counts and relative survival values are presented in Tables 1 to 3. Following treatment in the absence of S9 metabolic activation, using a 3-hour treatment time, no cells survived at dose levels between 156 and 5000 g/ml. Slight toxicity was observed over the remaining concentrations reducing survival to 74% of the concurrent negative control at 78.1 g/ml. Using a long treatment time, no cell survived at concentrations between 313 and 5000 g/ml, while at the next lower dose level (156 g/ml) survival was reduced to 44% of the negative control value. Following treatment in the presence of S9 metabolic activation, no cells survived at concentrations between 313 and 5000 g/ml. Dose-related toxicity was observed over the remaining dose levels, reducing survival to 8% of the negative control value at 156 g/ml. RTC Study No.: 52410 Page 18 6.3 6.3.1 Mutation assays Experimental design Two independent assays for mutation to trifluorothymidine resistance were performed using dose levels described in the following table: Assay No.: S9 1 - Treatment Time (hours) 3 Dose-level (g/ml) 125, 100, 80.0, 40.0, 20.0, 10.0 and 5.00 1 + 3 150, 100, 75.0, 37.5, 18.8 and 9.38, 2 - 24 160, 120, 80.0, 40.0, 20.0 and 10.0 2 + 3 120, 100, 83.3, 69.4 and 57.9 The results of the mutation assays are presented in Tables 4-9 (without S9) and 10-15 (with S9) and summarised in Tables 17-18. Untreated, solvent and positive control cultures were included in each mutation experiment in the absence and presence of S9 metabolism. From the data it can be seen that the mutant frequencies in the negative control cultures fell within the normal range (50-200 x 10-6 viable cells). The positive control chemicals induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). The cloning efficiencies at Day 2 in the untreated control cultures fell within the range of 65-120% and the control growth factor over 2 days fell within the range of 8 32 in both experiments. Results obtained are presented in the following table: Assay No.: S9 1 - Growth Factor 21 Cloning efficiency (%) 116 2 - 31 102 The study was accepted as valid. 6.3.2 Survival after treatment Results of relative survival are presented in Tables 4, 7, 10 and 13. Additional data of toxicity (RTG values) are presented in Tables 5, 8, 11 and 14. In the absence of S9 metabolic activation, using the 3-hour treatment time, very few cells survived at the highest dose level (125 g/ml). Marked toxicity was observed at the next lower concentration (100 g/ml) reducing survival to 23% of the concurrent negative control value. The relative total growth was reduced to 31% at the same concentration. In the second experiment, using a long treatment time, no cells survived at the highest concentration tested (160 g/ml), while at the next lower dose level (120 g/ml) the %RS and RTG values were reduced to 47% and 30% of the concurrent negative control value, respectively. RTC Study No.: 52410 Page 19 6.3.3 6.4 In the presence of S9 metabolic activation, in the first experiment, very few cells survived at the highest dose level (150 g/ml). Dose-related toxicity was observed over the remaining concentrations reducing the %RS and RTG values to 47% and 27% of the concurrent negative control value at 100 g/ml. In the second experiment, at the highest concentration tested (120 g/ml) no cells survived at the end of the phenotypic expression period, while at the next lower dose level (100 g/ml) the %RS and RTG values were reduced to 18% and 15% of the concurrent negative control value, respectively. It may be noted that in Experiment 1 in the presence of S9 metabolic activation the heterogeneity between replicate cultures for survival at the end of treatment, at the concentration of 100 and 75.0 g/ml was higher than usual. This has in no way affected the validity of the study. Mutation results In the absence of S9 metabolic activation, a statistically significant increase in mutant frequency was observed at the lowest concentration tested in the second experiment. However, the mutant frequency observed at this dose level was within the historical control range at RTC. In addition, no statistically significant dose-relationship was observed. Hence, the slight increases observed were considered to be attributable to a chance event not related to the action of 7850 and of no biological significance. In the presence of S9 metabolic activation, no statistically significant increases in mutant frequency were observed at any dose level, in any experiment. It may be noted that a higher than usual heterogeneity was observed for plating efficiency results between replicate cultures at the top dose tested in Experiment 2 in the absence of S9 metabolism. This has in no way affected the validity of the study. For the untreated, vehicle and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. The small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. Results are presented in Table 16. Good recovery of small colony mutants was observed following treatment with the positive controls. Osmolality and pH The pH values and osmolality of the post-treatment media were determined and results are presented in Table 19. The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium. RTC Study No.: 52410 Page 20 7. CONCLUSIONS It is concluded that 7850 does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions. RTC Study No.: 52410 Page 21 8. REFERENCES Robinson W.D. et al. in: Statistical Evaluation of Mutagenicity Test Data D.J. Kirkland (ed.) Cambridge University Press (1990). Cole J. et al. in: Report of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Part I revised D.J. Kirkland (ed.) United Kingdom Environmental Mutagen Society 1990. Cole J. and C.F. Arlett in: Mutagenicity testing - a practical approach Venitt S. and J.M. Parry (ed.) IRL Press Oxford. 1984. Clive D. et al. (1979) Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system Mutation Research 59 61-108. Clive D. et al. (1995) Consensus agreement regarding protocol issues discussed during the Mouse Lymphoma workshop: Portland. Oregon. May 7. 1994 Environ. Mol. Mutagen.. 25. 165-168. Moore M.M. et al. (2003) Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Test Workgroup Report - Plymouth, UK 2002. Mutation Research 540, 127-140 ICH Tripartite Harmonized Guideline (S2A) Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals Step 4 Final Draft - July 18. 1995. RTC Study No.: 52410 Page 22 9. TABLES 1 TO 19 RTC Study No.: 52410 Page 23 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 1 - CYTOTOXICITY TEST STUDY NO.: 52410 SOLVENT: Ethanol TREATMENT: 3 HOURS - WITHOUT S9 Dose-level Cells x 106 per ml Cell count (g/ml) Post treatment factor Total wells Plate counts# Cloning Survival efficiency (%) Ethanol 1% 0.65 19.5 0.56 39.1 0.49 78.1 0.56 156 0.06 313 0 625 0 1250 0 2500 0 5000 0 1.00 192 75 72 0.91 100 0.86 192 72 74 0.89 85 0.75 192 68 77 0.88 73 0.86 192 68 69 0.78 74 0.09 - 0 0.00 - 0 0.00 - 0 0.00 - 0 0.00 - 0 0.00 - 0 # = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival = Insufficient viable cells recovered after treatment incubation period RTC Study No.: 52410 Page 24 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 2 - CYTOTOXICITY TEST STUDY NO.: 52410 SOLVENT: Ethanol TREATMENT: 24 HOURS - WITHOUT S9 Dose-level Cells x 106 per ml Cell count (g/ml) Post treatment factor Total wells Plate counts# Cloning Survival efficiency (%) Ethanol 1% 0.54 19.5 0.56 39.1 0.48 78.1 0.43 156 0.23 313 0.01 625 0 1250 0 2500 0 5000 0 1.00 192 70 64 0.75 100 1.04 192 69 63 0.73 101 0.89 192 75 75 0.95 113 0.80 192 70 71 0.83 88 0.43 192 68 69 0.78 44 0.02 - 0 0.00 - 0 0.00 - 0 0.00 - 0 0.00 - 0 # = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival = Insufficient viable cells recovered after treatment incubation period RTC Study No.: 52410 Page 25 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 3 - CYTOTOXICITY TEST STUDY NO.: 52410 SOLVENT: Ethanol TREATMENT: 3 HOURS - WITH S9 Dose-level Cells x 106 per ml Cell count (g/ml) Post treatment factor Total wells Plate counts# Cloning efficiency Survival (%) Ethanol 1% 0.49 19.5 0.34 39.1 0.36 78.1 0.32 156 0.10 313 0.02 625 0 1250 0 2500 0 5000 0 1.00 192 65 60 0.66 100 0.69 192 70 68 0.79 84 0.73 192 62 63 0.66 73 0.65 192 60 69 0.70 69 0.20 192 32 36 0.27 8 0.04 - 0 0.00 - 0 0.00 - 0 0.00 - 0 0.00 - 0 # = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival = Insufficient viable cells recovered after treatment incubation period RTC Study No.: 52410 Page 26 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 4 - DAY 0 - SURVIVAL STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITHOUT S9 Dose-level Cells x 106 per ml Cell count (g/ml) Post treatment factor Total wells Plate counts# (Day 0) Cloning Survival efficiency (%) CM A 0.36 B 0.39 0.74 192 83 85 1.19 85 192 78 81 Ethanol A 0.47 1% B 0.55 1.00 192 74 78 1.03 100 192 82 76 5.00 A 0.44 B 0.42 0.84 192 71 75 0.86 70 192 68 73 10.0 A 0.39 B 0.33 0.70 192 80 84 1.27 86 192 82 88 20.0 A 0.43 B 0.33 0.75 192 76 81 1.05 77 192 76 80 40.0 A 0.50 B 0.45 0.92 192 67 68 0.80 72 192 73 70 80.0 A 0.34 B 0.31 0.64 192 77 77 1.10 68 192 80 84 100 A 0.21 B 0.19 0.39 192 70 66 0.61 23 192 50 54 125 A 0.06 B 0.05 0.10 - - 0 MMS 10.0 A 0.37 0.97 192 80 83 1.18 96 # CM A and B = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival = Culture medium = Replicate cultures = Insufficient viable cells recovered after treatment incubation period RTC Study No.: 52410 Page 27 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 5 - DAY 2 - TOTAL GROWTH AND VIABILITY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITHOUT S9 Dose-level (g/ml) Cells x 106 per ml Day 1 Cells x 106 per ml Day 2 Total wells Plate counts# ECffliocnieinngcy TSG (Day 2) Day 2 RTG CM A 1.11 0.69 192 80 88 1.16 15.3 121 B 1.00 0.86 192 79 76 Ethanol A 0.61 0.88 192 83 78 1.11 13.2 100 1% B 0.60 0.84 192 77 81 5.00 A 0.69 1.03 192 71 74 1.00 13.8 94 B 0.66 0.87 192 81 79 10.0 A 0.77 0.80 192 81 75 1.07 11.9 87 B 0.91 0.79 192 82 77 20.0 A 0.79 0.74 192 83 84 1.20 13.7 111 B 1.03 0.87 192 82 78 40.0 A 0.71 0.57 192 87 79 1.33 10.5 95 B 0.76 0.64 192 86 86 80.0 A 0.82 0.65 192 83 78 1.19 9.8 80 B 0.92 0.74 192 85 81 100 A 0.60 0.68 192 82 81 1.33 3.4 31 B 0.50 0.54 192 90 84 MMS 10.0 A 0.81 0.89 192 85 79 1.20 13.3 90 # CM A and B = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for viability = Culture medium = Replicate cultures RTC Study No.: 52410 Page 28 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 6 - MUTANT FREQUENCY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITHOUT S9 Dose-level (g/ml) Total wells Resistant mutants# after day 2 Mean mutant frequency (Per 106 viable cells) CM A 384 23 26 24 19 B 384 16 11 16 10 90.99 - Ethanol A 384 24 23 23 26 1% B 384 22 20 22 25 124.1 - 5.00 A 384 6 10 8 6 B 384 13 17 17 10 60.83 NS 10.0 A 384 10 9 7 11 B 384 15 6 11 13 52.62 NS 20.0 A 384 19 14 13 11 B 384 14 18 11 13 66.75 NS 40.0 A 384 18 16 17 16 B 384 12 14 17 13 65.80 NS 80.0 A 384 14 17 16 14 B 384 14 16 16 14 71.90 NS 100 A 384 15 15 16 22 B 384 22 20 15 15 68.81 NS MMS 10.0 A 384 56 55 48 50 326.5 Linear trend NS # A and B CM = Wells with clones/plate - 2 x 103 cells in each well of four 96-well plates plated for 5-TFT resistance = Replicate cultures = Culture medium RTC Study No.: 52410 Page 29 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 7 - DAY 0 - SURVIVAL STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 24 HOURS - WITHOUT S9 Dose-level Cells x 106 per ml Cell count (g/ml) Post treatment factor Total wells Plate counts# (Day 0) Cloning efficiency Survival (%) CM A 0.29 B 0.35 1.33 192 80 80 1.11 135 192 80 79 Ethanol A 0.24 1% B 0.24 1.00 192 81 75 1.10 100 192 82 80 10.0 A 0.31 B 0.20 1.05 192 74 79 0.95 90 192 75 72 20.0 A 0.21 B 0.33 1.11 192 80 76 0.96 97 192 75 70 40.0 A 0.27 B 0.22 1.01 192 80 82 1.00 92 192 74 70 80.0 A 0.17 B 0.21 0.78 192 80 80 0.98 70 192 71 73 120 A 0.13 B 0.12 0.52 192 70 79 1.00 47 192 77 80 160 A 0.01 0.01 - 0 B 0.00 - MMS A 0.28 5.00 0.88 192 68 70 0.79 62 # = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival CM = Culture medium A and B = Replicate cultures = Insufficient viable cells recovered after treatment incubation period RTC Study No.: 52410 Page 30 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 8 - DAY 2 - TOTAL GROWTH AND VIABILITY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 24 HOURS - WITHOUT S9 Dose-level Cells x 106 Cells x 106 Total Plate counts# Cloning per ml per ml wells (Day 2) Efficiency TSG (g/ml) Day 1 Day 2 Day 2 RTG CM A 1.19 1.18 192 70 69 1.02 65.5 149 B 1.32 0.82 192 81 84 Ethanol A 1.25 0.63 192 84 84 1.27 35.0 100 1% B 1.28 0.75 192 81 85 10.0 A 0.70 0.80 192 83 85 1.08 25.2 61 B 0.62 1.04 192 70 73 20.0 A 1.23 0.80 192 76 80 1.07 34.8 84 B 0.81 0.79 192 77 82 40.0 A 0.78 0.84 192 81 79 1.17 26.1 69 B 0.95 0.65 192 81 84 80.0 A 0.91 0.66 192 74 76 1.04 22.2 52 B 1.09 0.64 192 79 82 120 A 0.42 0.65 192 81 76 1.53 8.9 30 B 0.37 0.70 192 93 91 MMS A 1.35 0.67 192 79 76 1.03 42.2 65 5.00 # CM A and B = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for viability = Culture medium = Replicate cultures RTC Study No.: 52410 Page 31 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 9 - MUTANT FREQUENCY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 24 HOURS - WITHOUT S9 Dose-level (g/ml) Total wells Resistant mutants# after day 2 Mean mutant frequency (Per 106 viable cells) CM A 384 12 24 14 16 B 384 8 5 11 15 66.59 - Ethanol A 384 27 27 15 20 1% B 384 17 17 11 12 82.74 - 10.0 A 384 30 28 30 34 B 384 20 16 22 16 142.0 * 20.0 A 384 14 17 15 18 B 384 27 25 18 21 105.1 NS 40.0 A 384 14 11 19 11 B 384 13 17 18 19 73.88 NS 80.0 A 384 18 13 9 14 B 384 19 19 14 15 82.61 NS 120 A 384 12 19 10 12 B 384 22 18 16 18 66.05 NS MMS A 384 59 53 45 54 5.00 381.8 - Linear trend NS # CM A and B NS * = Wells with clones/plate - 2 x 103 cells in each well of four 96-well plates plated for 5-TFT resistance = Culture medium = Replicate cultures = Not statistically significant = Statistically significant at P<5% RTC Study No.: 52410 Page 32 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 10 - DAY 0 - SURVIVAL STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITH S9 Dose-level Cells x 106 per ml Cell count (g/ml) Post treatment factor Total wells Plate counts# (Day 0) Cloning efficiency Survival (%) CM A 0.25 B 0.25 1.04 192 75 76 1.16 133 192 86 87 Ethanol A 0.18 1% B 0.30 1.00 192 74 79 0.91 100 192 68 73 9.38 A 0.27 B 0.26 1.10 192 72 77 1.01 124 192 77 82 18.8 A 0.23 B 0.26 1.01 192 74 76 0.91 101 192 72 72 37.5 A 0.20 B 0.29 1.01 192 72 75 0.79 88 192 67 61 75.0 A 0.10 B 0.20 0.63 192 89 86 1.05 72 192 67 70 100 A 0.18 B 0.11 0.60 192 53 57 0.70 47 192 75 73 150 A 0.04 0.17 - 0 B 0.04 - B(a)P A 0.38 2.00 1.50 192 35 41 0.31 41 # CM A and B = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival = Culture medium = Replicate cultures = Insufficient viable cells recovered after treatment incubation period RTC Study No.: 52410 Page 33 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 11 - DAY 2 - TOTAL GROWTH AND VIABILITY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITH S9 Dose-level Cells x 106 Cells x 106 Total Plate counts# Cloning per ml per ml wells (Day 2) Efficiency TSG (g/ml) Day 1 Day 2 Day 2 RTG CM A 0.84 0.62 192 81 78 1.23 7.3 171 B 1.00 0.65 192 85 85 Ethanol A 0.32 1.06 192 74 77 1.07 4.9 100 1% B 0.67 0.67 192 79 84 9.38 A 0.77 0.76 192 81 74 1.01 8.0 155 B 0.73 0.86 192 78 75 18.8 A 0.66 0.75 192 81 80 1.25 5.5 131 B 0.58 0.71 192 86 84 37.5 A 0.74 0.80 192 81 80 1.12 6.6 141 B 0.58 0.87 192 80 79 75.0 A 0.44 0.76 192 86 78 1.06 3.1 63 B 0.83 0.55 192 74 74 100 A 0.18 0.64 192 87 85 1.29 1.1 27 B 0.24 0.82 192 84 78 B(a)P 2.00 # CM A and B A 0.41 1.18 192 48 47 0.43 9.2 44 = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for viability = Culture medium = Replicate cultures RTC Study No.: 52410 Page 34 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 12 - MUTANT FREQUENCY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITH S9 Dose-level (g/ml) Total wells Resistant mutants# after day 2 Mean mutant frequency (Per 106 viable cells) CM A 384 17 14 12 12 B 384 10 14 20 17 Ethanol A 384 13 9 9 17 1% B 384 24 13 19 21 9.38 A 384 15 17 9 19 B 384 15 14 14 14 18.8 A 384 19 25 26 20 B 384 24 22 28 13 37.5 A 384 12 14 8 14 B 384 19 25 14 25 75.0 A 384 20 14 11 18 B 384 14 10 9 11 100 A 384 14 20 17 12 B 384 19 21 16 23 67.41 - 83.49 - 81.62 NS 105.8 NS 83.50 NS 71.70 NS 80.23 NS B(a)P A 384 69 60 59 48 2.00 Linear trend 1117 - NS # CM A and B NS = Wells with clones/plate - 2 x 103 cells in each well of four 96-well plates plated for 5-TFT resistance = Culture medium = Replicate cultures = Not statistically significant RTC Study No.: 52410 Page 35 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 13 - DAY 0 - SURVIVAL STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 3 HOURS - WITH S9 Dose-level Cells x 106 per ml Cell count (g/ml) Post treatment factor Total wells Plate counts# (Day 0) Cloning efficiency Survival (%) CM A 0.47 B 0.49 1.06 192 71 75 0.91 111 192 74 75 Ethanol A 0.46 1% B 0.44 1.00 192 73 73 0.87 100 192 70 72 57.9 A 0.25 B 0.36 0.68 192 67 69 0.79 62 192 71 68 69.4 A 0.30 B 0.35 0.72 192 52 59 0.52 43 192 55 51 83.3 A 0.26 B 0.23 0.55 192 44 46 0.46 29 192 57 53 100 A 0.13 B 0.21 0.37 192 47 50 0.42 18 192 47 44 120 A 0.17 B 0.13 0.32 192 16 26 0.21 8 192 38 31 B(a)P A 0.38 2.00 0.54 192 57 53 0.53 31 # CM A and B = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival = Culture medium = Replicate cultures RTC Study No.: 52410 Page 36 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 14 - DAY 2 - TOTAL GROWTH AND VIABILITY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 3 HOURS - WITH S9 Dose-level Cells x 106 Cells x 106 Total Plate counts# Cloning per ml per ml wells (Day 2) Efficiency TSG (g/ml) Day 1 Day 2 Day 2 RTG CM A 0.60 B 0.57 Ethanol A 0.43 1% B 0.52 57.9 A 0.60 B 0.59 69.4 A 0.37 B 0.32 83.3 A 0.29 B 0.35 100 A 0.15 B 0.17 120 A 0.03 B 0.04 B(a)P A 0.43 2.00 0.90 192 77 71 0.86 13.6 130 1.04 192 70 69 0.71 192 82 82 1.16 7.8 100 0.75 192 78 82 0.67 192 76 81 1.05 5.7 66 0.60 192 78 77 0.72 192 76 79 1.06 3.9 45 0.68 192 78 80 0.70 192 80 81 1.14 2.4 30 0.52 192 82 79 0.55 192 77 83 1.02 1.3 15 0.93 192 74 74 - 0 0 - 0.66 192 76 69 0.88 5.4 40 # CM A and B = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for viability = Culture medium = Replicate cultures = Insufficient viable cells recovered at Day 1 RTC Study No.: 52410 Page 37 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 15 - MUTANT FREQUENCY STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 3 HOURS - WITH S9 Dose-level (g/ml) Total wells Resistant mutants# after day 2 Mean mutant frequency (Per 106 viable cells) CM A 384 11 8 17 16 B 384 8 11 11 10 74.19 - Ethanol A 384 23 16 16 20 1% B 384 22 19 16 16 92.26 - 57.9 A 384 9 13 22 16 B 384 17 14 17 9 78.99 NS 69.4 A 384 22 11 17 12 B 384 15 17 22 18 90.87 NS 83.3 A 384 23 11 21 11 B 384 16 21 13 19 84.81 NS 100 A 384 18 16 15 11 B 384 13 13 12 14 77.85 NS B(a)P A 384 75 73 72 76 2.00 837.5 - Linear trend NS # CM A and B NS = Wells with clones/plate - 2 x 103 cells in each well of four 96-well plates plated for 5-TFT resistance = Culture medium = Replicate cultures = Not statistically significant RTC Study No.: 52410 Page 38 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 16 - SMALL AND LARGE COLONY MUTANT FREQUENCIES STUDY NO.: 52410 EXPERIMENT NO.: 1 Dose-level g/ml S9 Mutant frequency Small colony Large colony Proportion small colony mutants CM - 37.2 49.7 0.43 Ethanol 1% - 65.5 50.9 0.56 MMS 10.0 - 183 95.7 0.66 CM + 31.2 34.1 0.48 Ethanol 1% + 33.6 47.6 0.41 B(a)P 2.00 + 417 484 0.46 EXPERIMENT NO.: 2 Dose-level S9 g/ml CM - Ethanol 1% - MMS 5.00 - CM + Ethanol 1% + B(a)P 2.00 + Mutant frequency Small colony Large colony 25.9 38.6 36.4 43.2 152 169 35.9 36.7 40.0 48.0 289 296 Proportion small colony mutants 0.40 0.46 0.47 0.49 0.45 0.49 = Figures displayed are mutation frequencies per 106 viable cells RTC Study No.: 52410 Page 39 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 17 - SUMMARY TABLE STUDY NO.: 52410 SOLVENT: Ethanol Dose-level (g/ml) %RS MAIN ASSAY 1 - WITHOUT S9 (Treatment time: 3 hours) RTG MF P Proportion small colony mutants CM 85 121 90.99 - 0.43 Ethanol 1% 100 100 124.1 - 0.56 5.00 70 94 60.83 NS - 10.0 86 87 52.62 NS - 20.0 77 111 66.75 NS - 40.0 72 95 65.80 NS - 80.0 68 80 71.90 NS - 100 23 31 68.81 NS - 125 0 - - MMS 10.0 96 90 326.5 - 0.66 Linear trend NS Dose-level (g/ml) %RS MAIN ASSAY 2 - WITHOUT S9 (Treatment time: 24 hours) RTG MF P Proportion small colony mutants CM 135 149 66.59 - 0.46 Ethanol 1% 100 100 82.74 - 0.40 10.0 90 61 142.0 * - 20.0 97 84 105.1 NS - 40.0 92 69 73.88 NS - 80.0 70 52 82.61 NS - 120 47 30 66.05 NS - 160 0 - - MMS 5.00 62 65 381.18 - 0.47 Linear trend NS =Figures displayed are mutation frequencies per million surviving cells NS = Not statistically significant = Insufficient viable cells recovered after treatment incubation period * = Statistically significant at p < 5% RTC Study No.: 52410 Page 40 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 18 - SUMMARY TABLE STUDY NO.: 52410 SOLVENT: Ethanol Dose-level (g/ml) %RS MAIN ASSAY 1 - WITH S9 (Treatment time: 3 hours) RTG MF P Proportion small colony mutants CM 133 171 67.41 - 0.48 Ethanol 1% 100 100 83.49 - 0.41 9.38 124 155 81.62 NS - 18.8 101 131 105.8 NS - 37.5 88 141 83.50 NS - 75.0 72 63 71.70 NS - 100 47 27 80.23 NS - 150 0 - B(a)P 2.00 41 44 1117 - 0.46 Linear trend NS Dose-level (g/ml) %RS MAIN ASSAY 2 - WITH S9 (Treatment time: 3 hours) RTG MF P Proportion small colony mutants CM 111 130 74.19 - 0.49 Ethanol 1% 100 100 92.26 - 0.45 57.9 62 66 78.99 NS - 69.4 43 45 90.87 NS - 83.3 29 30 84.81 NS - 100 18 15 77.85 NS - 120 8 - - B(a)P 2.00 31 40 837.5 - 0.49 Linear trend NS = Figures displayed are mutation frequencies per million surviving cells NS = Not statistically significant = Insufficient viable cells recovered after treatment incubation period = Insufficient viable cells recovered at Day 1 RTC Study No.: 52410 Page 41 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 19 - pH AND OSMOLALITY OF TREATMENT MEDIUM STUDY NO.: 52410 EXPERIMENT NO.: 1 SOLVENT: Ethanol Dose-level (g/ml) WITHOUT S9 pH mOsm/kg Dose-level (g/ml) WITH S9 pH mOsm/kg Culture medium A 7.63 281 B 7.64 281 Culture medium A 7.52 289 B 7.53 289 Ethanol 1% A 7.68 462 B 7.63 455 Ethanol 1% A 7.47 475 B 7.51 465 5.00 A 7.69 465 B 7.72 459 9.38 A 7.53 474 B 7.53 463 10.0 A 7.71 484 B 7.64 461 18.8 A 7.42 473 B 7.48 466 20.0 A 7.65 463 B 7.63 458 37.5 A 7.41 473 B 7.39 467 40.0 A 7.65 462 B 7.63 458 75.0 A 7.40 475 B 7.46 465 80.0 A 7.67 460 B 7.66 454 100 A 7.44 470 B 7.46 466 100 A 7.69 459 B 7.61 454 150 A 7.42 471 B 7.52 463 125 A 7.68 463 B 7.74 453 The solvent, ethanol, causes an increase in the apparent osmolality of the treatment solution, since it depresses the freezing point. However, since it passes freely in and out of cells, this increase is only apparent. A small but real increase in osmotic pressure may be caused only during the period of equilibration after the addition of the solvent. The increase due to the solvent (approx. 150 mOsm/kg) can therefore be subtracted from the treatment medium values, to assess the effects of the test substance. Since the test item displaces the solvent in the test item solution, lower apparent osmotic pressures may be observed at higher dose-levels. RTC Study No.: 52410 Page 42 10. APPENDIX I - Statistical analysis SYMBOLS USED IN THE STATISTICAL ANALYSIS Hs Survival heterogeneity factor Hv Viability heterogeneity factor Hm Mutation heterogeneity factor LMF Log mutant frequency W Weight for regression V Variance Di LMF treatment- LMF control Var(d) Variance of d b2 /Var(b) Test for linear trend/slope b and its variance Var(b) E Extention NA Not applicable RTC Study No.: 52410 Page 43 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 1 - CONSISTENCY TEST STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITHOUT METABOLIC ACTIVATION Dose-level (g/ml) Test for consistency between plates Survival Viability A B A B Mutants A B CM Ethanol 1% 5.00 10.0 20.0 40.0 80.0 100 0.190 0.505 0.457 0.669 0.874 0.025 0.000 0.403 0.329 1.287 0.668 1.848 0.547 0.247 0.669 0.336 3.048 0.962 0.254 1.231 0.046 2.847 0.962 0.041 0.301 0.572 0.150 0.915 0.600 0.000 0.712 2.087 1.487 0.333 1.591 1.047 2.864 0.199 0.526 2.430 2.692 0.746 2.864 4.506 2.174 1.171 0.316 2.598 A and B = Replicate culture Dose-level (g/ml) Test for overall consistency Survival (Hs) Viability (Hv) Ethanol 1% 5.00 10.0 20.0 40.0 80.0 100 0.603 0.345 0.828 0.017 0.834 1.830 11.378 0.167 3.586 0.159 1.009 0.889 0.515 13.190 H experiment 2.262 2.788 Current heterogeneity 1.801 1.705 Updated heterogeneity 1.824 1.759 Mutants (Hm) 0.349 9.450 0.874 0.010 1.171 0.010 0.140 1.715 1.771 1.768 RTC Study No.: 52410 Page 44 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 2 - CONSISTENCY TEST STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 24 HOURS - WITHOUT METABOLIC ACTIVATION Dose-level (g/ml) Test for consistency between plates Survival Viability A B A B Mutants A B CM Ethanol 1% 10.0 20.0 40.0 80.0 120 0.000 1.231 0.804 0.547 0.158 0.000 2.427 0.037 0.158 0.261 0.704 0.444 0.111 0.314 0.026 0.000 0.190 0.547 0.150 0.122 0.874 0.388 0.712 0.247 0.915 0.388 0.346 0.522 1.228 6.011 0.913 0.750 3.629 3.534 4.093 1.424 2.534 1.808 2.808 1.501 1.501 1.272 A and B = Replicate culture Dose-level (g/ml) Test for overall consistency Survival (Hs) Viability (Hv) Ethanol 1% 10.0 20.0 40.0 80.0 120 H experiment 0.659 0.549 1.860 5.213 4.042 1.030 2.225 0.092 10.571 0.159 0.501 2.047 (19.091) 2.674 Current heterogeneity 1.801 1.581 Updated heterogeneity 1.822 1.636 Values in parentheses are not included in the calculation of H experiment Mutants (Hm) 8.660 15.783 5.892 1.403 1.658 4.160 6.260 2.347 2.543 RTC Study No.: 52410 Page 45 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 3 - CONSISTENCY TEST STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT: 3 HOURS - WITH METABOLIC ACTIVATION Dose-level (g/ml) Test for consistency between plates Survival Viability A B A B Mutants A B CM Ethanol 1% 9.38 18.8 37.5 75 100 0.031 0.804 0.749 0.122 0.261 0.581 0.341 0.058 0.668 0.915 0.000 0.844 0.229 0.118 0.329 0.279 1.640 0.038 0.038 2.676 0.223 0.000 1.015 0.290 0.205 0.037 0.000 1.422 1.422 4.190 4.425 2.148 2.286 3.703 2.791 4.268 4.207 0.062 7.178 5.211 1.437 1.705 A and B = Replicate culture Dose-level (g/ml) Test for overall consistency Survival (Hs) Viability (Hv) Ethanol 1% 9.38 18.8 37.5 75 100 2.090 1.640 0.522 4.625 (24.684) (17.057) 2.516 0.066 1.773 0.075 4.376 2.299 Mutants (Hm) 8.036 0.091 0.066 11.274 3.920 2.212 H experiment 3.688 1.851 Current heterogeneity 1.563 1.564 Updated heterogeneity 1.669 1.578 Values in parentheses are not included in the calculation of H experiment 4.266 2.186 2.290 RTC Study No.: 52410 Page 46 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 4 - CONSISTENCY TEST STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 2 TREATMENT: 3 HOURS - WITH METABOLIC ACTIVATION Dose-level (g/ml) Test for consistency between plates Survival Viability A B A B Mutants A B CM Ethanol 1% 57.9 69.4 83.3 100 120 0.457 0.000 0.101 1.046 0.084 0.188 3.048 0.030 0.108 0.235 0.337 0.341 0.188 1.109 1.061 0.000 0.874 0.301 0.038 1.350 0.026 0.600 0.033 0.143 0.346 0.000 4.804 2.303 7.111 5.924 9.002 2.054 0.670 1.674 3.523 1.778 2.597 0.178 A and B = Replicate culture Dose-level (g/ml) Ethanol 1% 57.9 69.4 83.3 100 120 H experiment Current heterogeneity Updated heterogeneity Test for overall consistency Survival (Hs) Viability (Hv) 0.222 0.115 0.265 4.174 0.375 9.238 2.074 1.669 1.689 0.316 0.068 0.156 0.000 2.362 0.580 1.578 1.528 Values in parentheses are not included in the calculation of H experiment Mutants (Hm) 0.033 0.091 0.904 0.081 0.669 0.356 2.290 2.193 RTC Study No.: 52410 Page 47 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 5 - MUTATION ASSAY - WITHOUT METABOLIC ACTIVATION STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 Dose-level (g/ml) LMF Ethanol 1% 5.00 10.0 20.0 40.0 80.0 100 -8.994 -9.707 -9.852 -9.615 -9.629 -9.540 -9.584 W 3.876E+09 9.855E+09 1.259E+10 9.801E+09 1.055E+10 8.845E+09 1.029E+10 TREATMENT TIME: 3 HOURS V 0.0167 0.0274 0.0287 0.0229 0.0219 0.0219 0.0205 Di 0.000 -0.713 -0.858 -0.620 -0.635 -0.546 -0.590 Var(d) NA 0.044 0.045 0.040 0.039 0.039 0.037 D2/Var(d) NA - Test for linear trend EXPERIMENT NO.: 2 Dose-level (g/ml) LMF Slope (b) Var (b) B2/Var(b) W : 1.94E-08 : 1.18E-14 : 0.03 TREATMENT TIME: 24 HOURS V Di Var(d) D2/Var(d) Ethanol 1% 10.0 20.0 40.0 80.0 120 -9.400 -8.860 -9.161 -9.513 -9.401 -9.625 5.760E+09 2.410E+09 3.765E+09 6.393E+09 5.102E+09 8.123E+09 0.0254 0.0206 0.0241 0.0287 0.0287 0.0282 0.000 0.540 0.239 -0.113 -0.002 -0.225 Test for linear trend Slope (b) Var (b) B2/Var(b) : -1.49E-07 : 1.22E-14 : - - = Not calculated for negative Di or Slope(b) values NA 0.046 0.049 0.054 0.054 0.054 NA 6.35E+00 1.15E+00 - RTC Study No.: 52410 Page 48 METHOD) 7850: MUTATION IN L5178Y TK+/- MOUSE LYMPHOMA CELLS (FLUCTUATION TABLE 6 - MUTATION ASSAY - WITH METABOLIC ACTIVATION STUDY NO.: 52410 SOLVENT: Ethanol EXPERIMENT NO.: 1 TREATMENT TIME: 3 HOURS Dose-level (g/ml) LMF W V Di Var(d) D2/Var(d) Ethanol 1% 9.38 18.80 37.50 75.00 100.00 -9.391 -9.413 -9.154 -9.391 -9.543 -9.431 5.800E+09 5.781E+09 4.565E+09 5.992E+09 6.997E+09 6.817E+09 0.0247 0.0260 0.0196 0.0239 0.0278 0.0228 0.000 -0.023 0.237 0.000 -0.152 -0.040 NA 0.051 0.044 0.049 0.053 0.048 NA - 1.27E+00 3.02E-07 - Test for linear trend EXPERIMENT NO.: 2 Dose-level (g/ml) LMF Slope (b) Var (b) B2/Var(b) W : -1.22E-07 : 2.02E-14 : - TREATMENT TIME: 3 HOURS V Di Var(d) D2/Var(d) Ethanol 1% 57.90 69.40 83.30 100.00 -9.291 -9.446 -9.306 -9.375 -9.461 5.539E+09 6.395E+09 5.339E+09 6.146E+09 6.373E+09 0.0212 0.0251 0.0227 0.0226 0.0259 0.000 -0.155 -0.015 -0.084 -0.170 Test for linear trend Slope (b) Var (b) B2/Var(b) : -1.03E-08 : 1.86E-14 : - - = Not calculated for negative Di or Slope(b) values NA NA 0.046 - 0.044 - 0.044 - 0.047 - RTC Study No.: 52410 Page 49 11. APPENDIX II - Historical data HISTORICAL DATA OF NEGATIVE/SOLVENT AND POSITIVE CONTROLS (Mutation frequencies per million surviving cells) WITHOUT METABOLIC ACTIVATION (3 hour treatment) Negative Positive control control WITH METABOLIC ACTIVATION (3 hour treatment) Negative Positive control control WITHOUT METABOLIC ACTIVATION (24 hour treatment) Negative Positive control control Mean Value 90.2 467 93.9 648 87.8 566 Standard deviation 30.1 150 31.8 254 32.1 190 Upper confidence limit (P<1%) 168 NC 176 NC 171 NC 51 76 Observed range 44.9 - 53.4 - 217 278 - 933 n 76 117 117 43 43 NC = Not calculated n = number of experiment RTC Study No.: 52410 Page 50 12. APPENDIX III - Certificate of analysis RTC Study No.: 52410 Page 51 G) SOLVAY SOLEXIS C.D.C. 0316 Commessa N751 Centro Ricerche e Svilup Viale Lombardia, 20 20021 BoHate (Ml) Richiesta analisi Numero: Risposta Numero: Richiedente Pag. Data 23/11/2004 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 Risultati Si allega spettro 19F-NMR con caratterizzazione del prodotto Si nota la presenza di piccolissime quantita di estere Distribuzione Riferimenti 11542 RTC Study No.: 52410 Page52  :.:i ....... "Tj (t, t',: 11-a_ge E''t.t(1B'5a_li,2jtJN.. ,;.PE ex2 s2pL1l SAHl?LE: t,tb ?QV 0 200-1 1:.1Q,i"t!tm.t ,1iceto-.u!I filc /11p,ce/mFin11 -LKWSO" -_:J22:J.OICM1_.fid: AC'OUISI'IioN afr<J 2El2, 1:16 to rHI ., o.so 15110 . 'lS-1ilG:ll fb H-00 rn . ., t-pw.r 4.,5 dl LD!lO tof nt . .a"loc 9:lin u 2400 .0 l0ll0 1ao l'lOt uc:cd FUI.GS ,.dp y IHS&L.A"! ,. 45Lii,<i "l' 3s,:,rJ2. . 0 . lo h.:1m1 1513.IJl ,. -4.ill-5''1 .d,l 2.2965,,l :-p -S!i519.8 .,.,. ib u:o.n0s DEC, i:; \!T. dfrq d :ii9.9. {til1- dpw1: Q dof 0 dflll .nnn dmnt e daif 2(!(1- rRCCEii5-SJ:HG lb U.1f:ld "ftfil"' 2M -I pxo,c 1 fn 131G?2 =-i :.ic-.11p 1,1b!1 'lffit .k"HCt p.1;0-Cplot IS=484 FLK7850_32230N_00l. jpeg NOL.WE!GHT = 5S1 EQUIV. Wl:IGHl = 55 l Cl=l.O "/. CJ. C2=!J.1 "/, C3 "-0CF20-" "-OCF ('CF-) 0-" -oCF3 = -OCFCF3COOH = -OCF2CL = -OCF lCE'1j CF2CL = = -OCF-2.CF (CF:l) CL = -OCF2COOH 3.3 o. 8 1. l 15.S 29. 7 49.2 IL.o Page FLK78.S0_32230N_ PFPE Pu l-s.e Se.qumn,c;:;e: s2pu1 I I t -30 '--r-' 0.32 -40 ._....., -0 .oo so 1, 74 -GO -70 -0.19 -80 -90 120 .oo lI I -100 3 I J I -110 -120 o. 51 -130 -140 16. 50 l I I J I I t -150 ppm 13. APPENDIX IV - Study Protocol RTC Study No.: 52410 Page 54 R E SEARC H TOX I C O LOGY C E NT R E ROMA Version No. : 05/l LT 7850 MUTATION IN L5178Y TIC'"" MOUSE LYMPHOMA CELLS (FLUCTUATION MEIBOD) Final Protocol prepared for SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 2002 1 Bollate (MI) Italy by RESEARCH TOXICOLOGY CENTRE S.p.A. Via Tito Sperl, 12 00040 Pomezia (Rome) Italy RTC Enquiry Number: 524 1 0 Cofflmarclal Office RfC S.o.A. Vja TilO Speri. l 2 00040 l=lomezia (:Romau r ITALY Tet: + 39.06.91 095.1 Fax: i- '39.06.91 0.5737 -e--ma.il: mkt@rtc.i.c W'.yw,rtc.11 RTC Study No. : 52410 - 1 of 1 8 - Head om- and Adminltntlon HTC S.p.A. Via TilO Si::ieri, 1 2 00040 t=>Drnezia (11:oma) - I TA l.V Tel..:. + 39.06.9 1 095. 1 Fa:ll'. + 39.06.912.2233 P.O. Sox 1 530i 00 1 43 - Roma f1,,1t Laurentino January 2006 ITTC S.p.A. ca0i1are sociafe EYro 5.1 6 4.000 C.C.LA.A. n 375376 Reg. Soc. Trib. di Roma n 2828172 Cod. Fisc.: 00653120584 Partila IVA; 009200 1 1 00 1 Page 55. Version No. : 05/l LT 7850 MUTATION IN L5178Y TI(1 MOUSE LYMPHOMA CELLS (FLUCTUATION METHOD) MANAGEMENT OF STUDY Scientific Director Head of Genetic and Cellular Toxicology Study Director Spon s or : SOLVAY SOLEXJS S.p.A. Viale Lombardia, 20 2002 1 Bollate (MI) Italy Monitor QUALITY ASSURANCE Quality Assurance Manager LOCATION OF STUDY The study will be performed at : Research Toxicology Centre S.p.A. (RTC) Via Tito Speri, 12 00040 Pomezia (Rome) 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: 524 1 0 RTC Study No. : 5241 0 - 2 of l 8 - January 2006 Page 56 Version No.: 05/ILT MUTATION IN L5178Y TK" MOUSE LYMPHOMA CELLS (FLUCTUATION METHOD) 1 . INTRODUCTION 1.1 Objective To assay the test item for the ability to induce mutations in L5 l 78Y TK"- mouse lymphoma cells cultured after in vitro treatment in the absence and presence of S9 metabolism. 1.2 Regulatory requiremeuts 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 1 1 February 2004; ENV/MC/CHEM(98)17 "OECD principles on Good Laboratory Practice - as revised in 1 997"; Decreto Legislativo no. 1 20 of27 January 1 992 and subsequent revisions. Jn addition, the study is designed to comply with the experimental methods indicated in the guidelines of: EEC Council Directive 2000/32, Annex 4E. OECD Guidelines for the testing ofchemicals No. 476 (Adopted July 1 997). ICH, Topic S2A, Genotoxicity: Specific Aspects of Regulatory Tests, Step 4 (Document, July 1995). !CH, Topic S2B, A Standard Battery for Genotoxicity Testing of Pharmaceuticals, Step 4 (Document, July 1997). 1.3 Priuciples ofthe method The mutation assay method used in this study is based on the identification of L5 l 78Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5 l 78Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 1 1 . Cells which are heterozygous at the TK locus (TK"") may undergo a single step forward mutation to the TK"- genotype in which little or no TK activity remains. RTC Enquiry Number: 52410 - 3 of l 8 - January 2006 RTC Study No.: 52410 Page 57 Version No.: 05/ILT The cells used, L5 l 78Y TK', are derived from one of the two clones derived from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5 178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al. 1 979) and is accepted by many regulatory authorities. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been proposed that small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells. According to this hypothesis, point mutations and deletions within the active allele (intragenic event) produce large colonies. The assay is performed in the following way: first the cytotoxicity of the test item is determined, and dose-levels are selected for the mutation assays. Two mutation assays are then performed. The cells are treated with the test item in the absence and presence of S9 metabolism, and are subcultured for a number of generations to allow the pheno(ypic expression of the induced mutations. At the end of the expression time the cells are seeded in selective medium (in which only mutant cells can grow) and non-selective medium (to determine the surviving proportion). In the first experiment, the cells are exposed to the test item for a short treatment time (3 hours). If negative or equivocal results are obtained in the first experiment without metabolic activation, a second experiment in the absence of S9 metabolism will be performed using a longer treatment time (24 hours). If negative or equivocal results are obtained in the first experiment with metabolic activation, a second experiment will be performed and a modified dose-range will be used to focus on the highest concentrations that could be tested. If, however, the first experiment produces a clear positive response, no further experiments will be undertaken. 2- TEST ITEM 2.1 It is the responsibility of the Sponsor to supply the test item, accompanied by analytical data confirming the identity, purity, stability, strength and composition of the item, the solubility and stability in the proposed vehicle and details of any known hazards to laboratory staff. The test item should be accompanied by a certificate of analysis. RTC Enquiry Number: 52410 RTC Study No.: 52410 - 4 of l8 - January 2006 Page 58 Version No.: 05/I LT 2.2 Approximately I year after the final report has been issued, remaining amounts of the test item will be destroyed by incineration. An aliquot of the item will be retained within the archives of the testing facility for a period of ten years after which it will be destroyed. 2.3 The test item identity is indicated on previous pages ofthis 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 work; solutions will be prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test item. Unless specified by the Sponsor, concentrations of solutions will be expressed in terms ofmaterial as received, and not ofactive constituents. Preferred solvents will be sterile distilled water, culture medium, DMSO, ethanol, acetone. Other solvents may be used as necessary. 2.8 No assay of the test item stability, nor its concentration and homogeneity in 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 AND METHODS 3.1 L5178Y TK" mouse lymphoma cells L5 l 78Y TK" mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 95 1 8). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals forthe absence ofmycoplasmal contamination. Permanent stocks of the L5 l 78Y TK'1 cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Cultures of the cells are grown in RPMI I 640 minimal medium supplemented with I 0% horse serum heat-inactivated at 56C for 20 minutes before use (Complete medium 1 0%). The incubations are at 37C in a 5% carbon dioxide atmosphere (! 00% nominal relative humidity). RTC Enquiry Number: 52410 RTC Study No. : 52410 - 5 of I S - January 2006 Page 59 3.2 Media Minimal medium A RPMI 1640 (IX) L-glutamine (200 mM) Sodium pyruvate (100 mM) Non-essential amino acids ( 1 OOX) Streptomycin sulphate 50.000 ill/ml Penicillin G 50.000 UI/ml F 68 Pluronic Minimal medium B RPMI 1 640 (IX) L-glutamine (200 mM) Sodium pyruvate ( 1 00 mM) Non-essential amino acids ( 1OOX) Streptomycin sulphate 50.000 ill/ml Penicillin G 50.000 ill/ml Complete medium (5%) Minimal medium A Horse serum (heat-inactivated) Complete medium (10%) Minimal medium A Horse serum (heat-inactivated) Complete medium A (20%) Minimal medium A Horse serum (heat-inactivated) Complete medium B (20%) Minimal medium B Horse serum (heat-inactivated) 5 16.1 ml 5.4 ml 6.0 ml 5.4 ml 1 . 1 ml 6.0 ml 522.1 ml 5.4 ml 6.0 ml 5.4 ml I . I ml 950 ml 50 ml 900 ml 100 ml 800 ml 200 ml 800 ml 200 ml Version No.: 05/ILT RTC Enquiry Number: 5 2410 RTC Study No.: 52410 - 6 of l8 - January 2006 Page 60 3.3 Preparation of S9 Mix Version No.: 05/lLT 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 beta-naphthoflavone (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 in the following proportions: S9 tissue fraction NADP (0.03 M) G-6-P (0.59 M) KC! (150 mM) Complete medium (5%) 0.408 ml 0.204 ml 0.204 ml 0.204 ml =8.= 98 m=l 1 0.0 ml Where it is necessary to change the composition of the S9 mix, all modifications will be indicated in the final report. 3.4 Control items Positive control treatments are included in every experiment. The positive control agents, methylmethanesulphonate (MMS), and benzo(a)pyrene (BP) are both obtained commercially, and characterised by their labelling. Fresh solutions of MMS in sterile distilled water are prepared for each day's work. Solutions of BP are prepared in dimethylsulphoxide and may be stored at -20C. 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. CYTOTOXICITY ASSAY 4.1 Experimental design A preliminary cytotoxicity test is undertaken in order to select appropriate dose levels for the mutation assays. In this test a wide range ofdose-levels ofthe test item, set at intervals of a factor of two, are used; cell cultures are treated using the same treatment conditions as the mutation assays, and the survival of the cells is subsequently determined. RTC Enquiry Number: 52410 RTC Study No.: 52410 - 7 of l 8 - January 2006 Page 61 Version No.: 05/lLT The test includes the following treatments: Solvent/vehicle controls The final concentration of organic solvents will not exceed 1 %. Test item The highest dose-level will be determined by the solubility of the test material, up to a maximum of 5 mg/ml. At the discretion of the Study Director, dose-levels where test item precipitation occurs may be employed. Treatments will be performed both in the absence and presence of S9 metabolism; a single culture will be used at each test point. Where it seems advisable, further test points may be included in the cytotoxicity test. 4.2 Test procedure The cultures will be prepared, and the treatment conducted using the methods described in Sections 6. 1, 6.2 and 6.3. The plates will be incubated for one to two weeks. Wells containing viable clones will be identified by eye using background illumination and counted. 4.3 Evaluation and selection of doses Percentage survival relative to the solvent controls will be calculated for each treatment. Dose-levels giving a predicted 1 0-20% survival will be estimated from the results obtained; the with and without S9 series may be considered separately, if appropriate. The estimated concentrations will be chosen as the highest dose-levels for the mutation assays. If the test item is not sufficiently toxic to reduce survival to 1 0-20%, the highest practicable dose-level (up to a maximum of 5 mg/ml) will be selected. A minimum of five dose-levels separated by a factor of two will be selected for the mutation assay. Where there is cytotoxicity, these concentrations should cover a range from the maximum to little or no toxicity. Selection of dose-levels for the second experiment will be based on the results obtained in the first experiment as described in Section 1 .3. 5. EXPERIMENTAL DESIGN (MUTATION ASSAY) Each experiment will include solvent/vehicle and positive controls and at least four analysable concentrations of the test item, tested in the absence and presence of S9 metabolising system. RTC Enquiry Number: 52410 RTC StudyNo.: 52410 - 8 of \8 - January 2006 Page 62 Version No.: 05/lLT Duplicate cultures will be prepared at each test point, with the exception of the positive controls which will be prepared in a single culture. Solvent/vehicle controls Treated with the maximum amount of solvent/vehicle used in any test item treatment. Positive controls Methylmethanesulphonate (experiments in the absence of S9 metabolism) or benzo(a)pyrene (experiments in the presence of S9 metabolism) will be used, both at the appropriate concentration to give a clear positive response. Test item The selection of test item dose-levels is described in the preceding section. Where it seems advisable, further test points or controls may be included in experiments. 6. lWUTATION ASSAY PROCEDURE 6.1 Preparation ofthe test system On the day of the experiment, sufficient number of cells will be used to prepare a cell suspension ( 1 x 10' cells/ml) in complete medium (3-hour treatment time: 5%; 24-hour treatment time: 10%). A common pool will be used to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes. 6.2 Treatment On the day ofthe experiment, treatment media will be prepared as follows: Without S9 metabolism - 3 hours treatment Cell suspension (JxlO' cells/ml) Complete medium (5%) Control or test item solution 1 0.0 ml 9.8 ml 0.2 ml 20.0 ml RTC Enquiry Number: 52410 RTC Study No.: 52410 - 9 of l8 - January 2006 Page 63 Without S9 metabolism - 24 hours treatment Version No.: 05/ILT Cell suspension (Ix1O' cells/ml) Complete medium ( 1 0%) Control or test item solution 3.0 ml 16.8 ml 0.2 ml 20.0 ml With S9 metabolism Cell suspension ( Ix10' cells/ml) S9 mix Control or test item solution 10.0 ml 9.8 ml 0.2 ml 20.0 ml Where it is necessary to adjust the composition of the treatment medium (for example when the culture medium is used as the solvent), the final composition used will be indicated in the final report. The cultures will be incubated at 37C. At the end of treatment time the cell cultures will be centrifuged and washed twice with Phosphate Buffered Saline (PBS). Fresh complete medium ( 10%) will be added and cell densities determined. The number of cells will be adjusted to give 2x10' cells/ml. The cultures will be incubated at 37C in a 5% CO, atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype. The pH and osmolality ofthe treatment solutions will be measured during the performance of one of the main experiments. 6.3 Determination of survival Following adjustment of the cell densities, samples of the cultures will be diluted to 8 cell/ml using complete medium A (20%). 0.2 ml ofeach diluted culture will be placed into each well of two 96-well plates. The plates will be incubated at 37C in a 5% CO, atmosphere (100% nominal relative humidity) for one to two weeks. After incubations, wells containing viable clones will be identified by eye using background illumination and counted. RTC Enquiry Number: 524 1 0 RTC Study No.: 52410 - !O of l8 - January 2006 Page 64 Version No.: 05/ILT 6.4 Determination of mutant frequency During the expression period (two days after treatment) the cell populations will be subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture will be determined and adjusted to give 2 X 1 0' cells/ml. The following two procedures will be carried out with each culture: (i) After dilution, the cell suspension in complete medium B (20%) will be supplemented with trifluorothymidine (final concentration 3.0 glml) and an estimated 2 X 1 0' cells will be plated in each well offour 96-well plates. Only TK" mutant colonies are able to grow in the presence of trifluorothymidine; these plates will be subsequently scored for the presence of mutants. (ii) After dilution, in complete medium A (20%), an estimated 1 .6 cells/well will be plated in each of two 96-well plates. These plates will be used to estimate Plating Efficiency (P.E.). 6.5 Incubation and scoring Plating efficiency and mutant plates will be incubated for one to two weeks to ensure adequate colony size. Plates prepared on any one day will receive the same period of incubation. After incubation, wells contammg viable clones will be identified by eye using background illumination and counted. Small colony and large colony mutant frequencies will be estimated for the solvent/vehicle and positive controls and for the doses oftest item showing positive results. The definition of the small and large colonies is as follows: I ) Size: Small: less than ofwell's diameter Large: more than ofwell's diameter 2) Morphology: Small: compact Large: diffused, totally or in periphery The positive control items should be capable of producing both small and large colonies in the L5 l 78Y TK" mouse lymphoma assay. RTC Enquiry Number: 52410 RTC Study No.: 52410 - 1 1 of l8 - January 2006 Page 65 7. REPORTING 7.1 Calculations 7. 1 . 1 Survival or viability Version No.: 05/lLT The following formula will be used to determine the plating efficiency (PE): PE where: -In (ys/ns) fs ys Number of empty wells (without clones) ns Total number ofwells fs Number of cells plated per well (I .6 cells/well) Plating efficiencies in test and control cultures will be compared to give the percentage relative survival. The survival calculation will take into account the reduction in post treatment cell-count as follows: % RS = % PE x cell count factor where cell count factor = treated post-treatment cell count control post-treatment cell count In order to aid toxicity data interpretation the relative total growth, expressed as a percentage of the concurrent solvent/vehicle control, will also be calculated. This is a product of the relative suspension growth (RSG) and the Day 2 relative plating efficiency (RPE), expressed as a percentage of the concurrent negative control, for each culture, as follows: RTG = RSG x RPE (Day 2) Relative suspension growth (RSG) is given by: [TSG(test) / TSG (control)] x 100 where TSG (total suspension growth) is calculated as follows: [post treatment cell count] TSG=: -------- ---- [pre treatment cell count] [Day I cell count] [2 X ]0'*] [Day 2 cell count] [2 X 1 05*] * Or appropriate cell concentration if lower RTC Enquily Number: 52410 RTC Study No.: 52410 - 12 of l8 - January 2006 Page 66 Version No.: 05/JLT 7.1 .2 Mutation frequency The following formula will be used to determine the mutation frequency (MF): MF [PE(mutant)/PE(viable)J x 1 0' Therefore: MF where: { -In (ym/nm) / -In (ys/ns) l X 10' frn fs ym Number of empty wells (mutant plates) nm Total number of wells (mutant plates) fm Number of cells plated per well (2xI 0' cell/well) ys Number of empty wells (viability plates) ns Total number of wells (viability plates) fs Number of cells plated per well (1.6 cells/well) Statistical analyses will be performed according to UKEMS guidelines (Robinson W.D., 1 990). 7.2 Evaluation For a test item to be considered mutagenic in this assay, it is required that: (i) The mutant frequency at one or more doses is statistically significantly greater than that of the solvent/vehicle control. (ii) There is a significant dose-relationship as indicated by the linear trend analysis. Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance. 7.3 Presentation of data The results will be presented in the form of tables which will show the individual plate counts in non-selective medium, the calculated plating efficiency, the individual plate counts in selective medium, the total number of mutant colonies and the calculated mutation frequency. Small and large colony mutant frequencies for negative and positive controls and for doses which show positive results will also be presented. RTC Enquiry Number: 52410 RTC StudyNo. : 52410 - 13 of l 8 - January 2006 Page 67 Version No.: 05/l LT 7.4 Reporting procedure A Draft Report will be supplied, and a Final Report issued subsequently to include any agreed changes or amendments. Ifany 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. 7.5 Final report The following information and data will be included in the final report: - name and address of the facility performing the study and the dates on which the study was initiated and completed; - objective and procedures stated in the approved protocol, including any approved changes to the original protocol; - data generated while conducting the study; - statistical methods employed for analysing the data; - 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; - the name and signature ofthe Study Director; - a summary of the data, an analysis of the data and a statement of the conclusions drawn for the analysis; - historical solvent/vehicle and positive control data with ranges, means and standard deviations; - QAU statement; - the location where all raw data, specimens and final report are to be stored. One original unbound, one copy bound and a PDF version will be supplied. RTC Enquiry Number: 52410 RTC Study No.: 52410 - 14 of l 8 - January 2006 Page 68 Version No.: 05/ILT 7.6 Records kept Full records will be maintained ofall aspects of study conduct, along with the results ofall measurements and observations. Prior to final archiving ofthe study data a full list will be prepared of all records associated with the study. 7.7 Archiving All raw data, records and documentation arising from this study and a copy of the final report consigned generated during the course of this study will be retained at RTC. Archiving will be provided for a period of 3 years after which the Sponsor will be contacted for instructions regarding despatch or disposal of the material. As a further option, archiving space can be rented for an additional time. The signed Final Protocol and the top copy of the Final Report will be despatched to and archived by the Sponsor. 8. STUDY CONDUCT 8.1 Langnage 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 oftest 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/she has reached and the reasoning which led to it. 8.3 Quality assurance The study is subjected to the procedure for quality assurance as defined by the relevant GLP regulations. Specifically: the protocol is inspected for compliance; procedures and data of the laboratories concerned will be inspected at intervals adequate to assure the integrity ofthe study; the final report is reviewed to ensure that it accurately describes the methods and relevant Standard Operating Procedures and that the results are in agreement with the raw data; periodic reports on these activities are made to management and the Study Director. RTC Enquiry Number: 524I 0 - 15 of l 8 - January 2006 RTC Study No.: 52410 Page 69 Version No.: 05/ILT All raw data pertaining to this study will be available for inspection by the study monitor (for scientific monitoring) or the Quality Assurance Unit of the Sponsor (compliance monitoring). 9. DEPARTURES FROM REGULATORY REQUIREMENTS Items which are the responsibility ofthe Sponsor are indicated in sections 2.1, 2.4, 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: 52410 RTC Study No.: 52410 - 16 of l 8 - January 2006 Page 70 10. REFERENCES Version No.: 05/JLT Amacher D.E. et al. ( I 980) Point mutations at the thymidine kinase locus in L5 l 78Y mouse lymphoma cells II. Test validation and interpretation Mutation Research 72, 447-474. Clive D. et al. (1979) Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system Mutation Research 59, 61-108. Cole J. et al. in: "Report ofUKEMS Sub-Committee on Guidelines for Mutagenicity Testing" Dean BJ. (ed.) United Kingdom Environmental Mutagen Society 1983. Cole J, Arlett C F, Green M H L, Lowe J and Muriel W (1983) A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells. Mutation Research 1 1 1 , 3 1 7-386. Cole J. and C.F. Arlett in: "Mutagenicity testing - a practical approach" Venitt S. and J.M. Parry (eds.) IRL Press, Oxford, I 984. Moore M M, Clive D, Rozier J C, Howard B E, Gail Batson A, Turner N T and Sawyer J (] 985) Analysis of TFT mutants of L5 l 78Y!TK" mouse lymphoma cells. Mutation Research 1 5 1 , 161-174. Moore M.M. et al. (2000) Mouse Lymphoma Thymidine Kinase Locus Gene Mutation Assay: International Workshop on Genotoxicity Test Procedures Workgroup Report Enviromental and Molecular Mutagenesis, 35 1 85-190 Moore M.M. et al. (2003) Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Test Workgroup Report - Plymouth, UK 2002. Mutation Research 540, 127-140 Robinson W D, Green M H L, Cole J, Garner R C, Healy M J R and Gatehouse D (1990) Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical Evaluation of Mutagenicity Test Data (Ed Kirkland D J) Cambridge University Press, pp 102-140. RTC Enquiry Number: 52410 RTC Study No. : 52410 - 17 of l8 - January 2006 Page 71 STUDY TITLE TEST FACILITY RTC ENQUlRY NO. TEST ITEM APPROVED BY Version No. : 05/lLT PROTOCOL APPROVAL PAGE MUTATION IN L5 1 78Y TK+1 MOUSE LYMPHOMA CELLS (FLUCTUATION METHOD) RESEARCH TOXICOLOGY CENTRE S.p.A. Via Tito Sperl, 12 00040 Pomezia (Rome) Italy 524 W RELEASED BY Date Technical & Operational Director SPONSOR SOLVAY SOLEXIS S.p.A. Viale Lombardia, 20 2002 1 Bollate (MI) Italy AUTHORISED BY SPONSOR Date Name and Title * Please print or type your name and company status below your signature RTC Enquiry Number: 524 1 0 RTC Study No. : 5241 0 - 1 8 of 1 8 - January 2006 Page 72