FINAL REPORT

Unpublished Work

Testing Facility Study No. Sponsor Report No. Work Request No. Service Code



### In Vitro Mammalian Chromosome Aberra tion Test Using Gas-phase Exposure

TESTING FACILITY:

SPONSOR:

STUDY COMPLETED ON 04 June 2010

Page 1 of 48Testing Facility Study No.

Page 3 2. PARTICIPATING PERSONNELTesting Facility Study No.

Page 6 TABLE OF CONTENTS

1. COMPLIANCE STATEMENT ...........................................................................................2 2. PARTICIPATING PERSONNEL .......................................................................................3 3. QUALITY ASSURANCE STATEMENT...........................................................................4 4. CERTIFICATION STATEMENT ......................................................................................5 LIST OF TABLES .........................................................................................................................8 LIST OF FIGURES .......................................................................................................................9 LIST OF APPENDICES .............................................................................................................10 5. STUDY INFORMATION...................................................................................................11 6. SUMMARY INFORMATION...........................................................................................12 7. INTRODUCTION ...............................................................................................................13 8. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS ..................................13 8.1. Control Atmosphere...................................................................................................13 8.2. Test Substance ............................................................................................................13 8.3. Positive Controls.........................................................................................................14 8.3.1. In the Absence of S9 Mix ............................................................................14 8.3.2. In the Presence of S9 Mix ...........................................................................14 8.4. Gaseous Positive Control ...........................................................................................14 9. EXPERIMENTAL PROCEDURES..................................................................................14 9.1. Test System .................................................................................................................14 9.1.1. Blood Sampling............................................................................................14 9.1.2. Culture Medium ..........................................................................................14 9.1.3. Lymphocyte Culture ...................................................................................15 9.2. S9 Mix..........................................................................................................................15 9.3. Stability and Chemical Analysis of Atmosphere Concentration............................15 9.4. Treatment and Test Atmosphere Generation..........................................................15 9.5. Medium Change (4-hour Treatment Only)..............................................................17 9.6. Harvesting and Metaphase Preparation ..................................................................17 9.7. Staining........................................................................................................................17Testing Facility Study No.

Page 7 9.8. Slide Examination.......................................................................................................17 9.8.1. Mitotic Index (MI).......................................................................................17 9.8.2. Selection of Slides for Detailed Examination............................................18 9.8.3. Detailed Examination for Chromosome Aberrations..............................18 10. EVALUATION AND INTERPRETATION OF RESULTS ...........................................19 11. QUALITY ASSURANCE ...................................................................................................19 12. PROTOCOL DEVIATIONS..............................................................................................20 13. ARCHIVES..........................................................................................................................20 14. RESULTS........................................................................................................................ .....20 14.1. Dose Selection .............................................................................................................20 14.2. Chromosome Aberrations .........................................................................................20 14.3. Incidental Observations.............................................................................................21 15. CONCLUSION....................................................................................................................21Testing Facility Study No.

Page 8 LIST OF TABLES

Table 1 Results and Statistical Analysis ............................................................................ 22Testing Facility Study No.

Page 9 LIST OF FIGURES

Figure 1 Historical Control Values ..................................................................................... 23Testing Facility Study No.

Page 10 LIST OF APPENDICES

Appendix 1 Certificate of Analysis .......................................................................................... 24 Appendix 2 Results for Individual Cultures ............................................................................. 26 Appendix 3 Protocol and Amendments.................................................................................... 28Testing Facility Study No.

Page 11 5. STUDY INFORMATION
Substance Tested : ï

ï (CAS Number)
Number : 2 Composition : (mole %)

Purity : See composition, above Physical Characteristics : Colorless gas Stability : The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Study Initiated/Completed : 08 March 2010/(see report cover page) Experimental Start/Termination : 11 March 2010/09 April 2010Testing Facility Study No.

Page 12 6. SUMMARY INFORMATION The purpose of this study was to evalua te the genotoxicity of using an in vitro chromosome aberration test. Human peripheral blood lymphocytes were stimulated into division in cult ure then treated with the gaseous test substance at a range of concentrations separate d by a 2-fold dose interval up to 50% v/v, (i.e., the maximum practical level not expected to result in anoxia). Cultures were treated for 4 hours in the absence and presence of rat S9 mix and for 21 hours in the absence; appropriate concurrent vehicle and positive controls were included for each treatment regime. Metaphases from cultures treated with the thr ee highest dose levels of test substance not producing excessive toxicity (tog ether with appropriate vehicle and selected positive control cultures) were subjected to detailed examinati on for the presence of chromosomal aberrations using light microscopy. Cultures treated with at levels up to 50% v/v did not show any st atistically significant increases in the incidence of aberrant metaphases in either the absence or presence of S9 mix. The positive control agents caused highly signifi cant increases in the proportion of cells with chromosome damage, confirming the sensitivity of the system and the effectiveness of the S9 mix.
It is concluded that did not show any evidence of genotoxicity in this in vitro test for induction of chromosome damage when tested in accordance with regulatory guidelinesTesting Facility Study No.

Page 13 7. INTRODUCTION The purpose of this study was to evalua te the genotoxicity of using the in vitro chromosome aberration test. Primary cultures of human peripheral lymphocytes are preferred for this type of study because of their stable chromosome number and structure (karyotype) compared with cell lines, and their low and stable background rate of aberrations. In addition, human cells are generally the most relevant for risk assessment. The present study was initiated on 08 March 2010 (date of appr oval of the protocol by the Study Director) and completed on the date of the St udy Director approval of this report (refer to the appropriate signature page). The experimental phase of the study was performed between 11 March 2010 (date of first treat ment of cultures with the te st substance) and 09 April 2010 (last date of collection of re sults directly from the study).

8. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS
8.1. Control Atmosphere Ambient laboratory air was used to treat negative/ vehicle control tubes and as the diluent for the test substance.

8.2. Test Substance Identity CAS number Chemical name Molecular weight Action (intended use) Industrial chemical Lot number Expiry 30 June 2011 Description Colorless gas under normal room conditions, boiling point 7.5°C Storage conditions Kept in a pressurized cylinder in a ventilated area at ambient/controlled room temperature Supplier Sponsor Purity/Correction Assumed to be 100%; all c oncentrations throughout this report are expressed in terms of material as is The above information relating to the nature of the test substance is the responsibility of the Sponsor (see Appendix 1 for Certificate of Analysis).Testing Facility Study No.

Page 14 8.3. Positive Controls
8.3.1. In the Absence of S9 Mix Identity Mitomycin C (MMC) CAS number 50-07-7 Vehicle Purified water (sterile water for irrigation USP) Supplier Sigma

8.3.2. In the Presence of S9 Mix Identity Cyclophosphamide monohydrate (cyclophosphamide, CP) CAS number 6055-19-2 Vehicle Purified water (sterile water for irrigation USP) Supplier Sigma

8.4. Gaseous Positive Control Identity Ethylene oxide (ETOX) CAS number 75-21-8 Vehicle Ambient laboratory air Supplier Sigma Full details of positive controls including expiry da te, lot number, appearance, storage, copies of certificates of analysis (whe re available) and formulati on are retained as raw data.

9. EXPERIMENTAL PROCEDURES
9.1. Test System
9.1.1. Blood Sampling A peripheral blood sample was taken from a h ealthy, non-smoking, male donor by venipuncture. The blood sample was taken directly into blood co llecting tubes containing sodium heparin then held at room temperature for less th an 2 hours prior to culture initiation.

9.1.2. Culture Medium Complete RPMI 1640 medium was prepared by supplementing RPMI 1640 with the following filter-sterilized components: 10% (v/v) fetal calf serum, 50 μg gentamycin per mL and 4 units heparin per mL.Testing Facility Study No.

Page 15 9.1.3. Lymphocyte Culture Whole blood was mixed with medium (0.4 mL blood per 4.6 mL medium) and phytohemagglutinin (1 mL per 49 mL diluted blood) was added to stimulate lymphocyte division. Aliquots of 5 mL of cell suspension were dispensed into flat-sid ed culture tubes then incubated at approximately 37°C in a hu midified atmosphere containing 5% CO 2 .
9.2. S9 Mix The S9 mix, used as a model of intact mammalia n metabolism, was prepared on the day of use and contained 10% v/v S9 fraction (phe nobarbital/5,6-benzoflavone induced male Sprague-Dawley rat liver fraction supplied by Molt oxô) and the following sterile cofactors: 8 mM MgCl 2 , 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP. The S9 mix was kept in a refrigerator set to maintain 4°C or on ice until required. A copy of th e manufacturerís quality control certificate(s) for the S9 fraction is retained as raw data.

9.3. Stability and Chemical Analysis of Atmosphere Concentration Test atmosphere stability and achieved concentra tion were not assessed as part of this study. The positive control formulations were not subjected to analysis for safety reasons and because the biological response of the test system is considered to be the best measure of the appropriateness of the formulations.

9.4. Treatment and Test Atmosphere Generation Treatments were performed approximately 48 hours after culture in itiation. The cell suspensions to be treated with gaseous test substances were transferred to gas-tight tubes (24 mL glass anaerobic culture tubes supplied by Bellco Gla ss) and each tube was sealed with a gas impermeable, butyl rubber septum (injectable) stopper and an alum inum seal. Using a gas-tight syringe and needle, the appropria te volume of air was removed from each treatment tube, then the appropriate volume of treatment gas was added. For the cell suspensions to be treated with the routine positive controls, 10 μ L positive control per mL of culture were used throughout and added to the cultures in the original 5 mL flat-sided tubes. All the cultures were incubated at
ca. 37°C in a humidified atmosphere containing 5% v/v CO 2 . was tested over a wide range of dose leve ls (0.1 to 50.0% v/v) using all treatment regimes (4 hour treatment period in the absence and presence of S9 mix and a 21 hour treatment period in the absence of S9 mix) so that anal yzable cells would be available for at least three dose levels for each regime. Duplicate cu ltures were treated at each experimental point.Testing Facility Study No.

Page 16 Cultures tested in the absence of S9 mix were treated as indicated in the study design then returned to the incubator for either 4 or 21 hours. For cultures tested in the presence of S9 mix, 1 mL culture medium was removed and replaced w ith 1 mL of S9 mix; each culture was treated as indicated in the study design then re turned to the incubator for 4 hours.
Text Table 1 Study Design Number of cultures Dose number Material Exposure conc. % v/v 4 Hours (0S9) 4 Hours (+S9) 21 Hours (0S9) 0 Vehicle control

- 2 2 2 1 Test substance 0.1 2 2 2 2 0.2 2 2 2 3 0.4 2 2 2 4 0.8 2 2 2 5 1.6 2 2 2 6 3.2 2 2 2 7 6.4 2 2 2 8 12.8 2 2 2 9 25.6 2 2 2 10 50.0Ü 2 2 2 1 ETOX 0.05 2 2 2 2 1.0 2 2 2 3 2.0 2 2 2 1 MMC a 0.05 2 2 2 0.10 2 2 3 0.20 2 2 1 CP a 8.0 2 2 12 2 3 16 2 Ü The highest dose tested was the maximum practical level without resulting in anoxia a Standard positive controls dosed as solutions; in units of μg/mL

Note that the 21 hour incubati on (ëconfirmatory phaseí) is requi red by regulatory authorities where the initial phase does not show any indi cation of genotoxicity for the test substance; however, it is routinely performed at the same time as the initial phase to minimize potential delays in performance of the study.Testing Facility Study No.

Page 17 9.5. Medium Change (4-hour Treatment Only) After the 4-hour treatment, the appropriate gas- treated cultures were opened in a laminar flow hood to allow the gas to dissipate , and the cells were transferre d to flat-sided culture tubes (Nunc). All 4-hour treatment cultures were then centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and replaced with fresh complete medium. The cultures were returned to the incubator for another 17 hours of incubation before metaphase harvesting. For 21-hour treated cultures (i.e., cultures in th e confirmatory phase), no replacement of medium was performed.

9.6. Harvesting and Metaphase Preparation After colcemid addition at a final concentration of 0.1 μg per mL, all cultures were incubated for approximately two hours prior to harvesting. The addition of colcemid into gas-tight tubes for the 21 h 0S9 regime was done through a needle a nd syringe. After 2 h, the gas-treated cultures from the 21 h 0S9 regime were opened in a lami nar flow hood to allow th e gas to dissipate and the cells were transferred to flat-sided cultu re tubes (Nunc). Cultures were harvested by centrifugation at 1000 rpm for 5 mi n. The supernatant was dis carded and the cell pellet was resuspended in 0.075 M KCl (hypotonic solution) and incubated for approximately 12 min at
ca . 37°C. Fixative (3 vol methanol:1 vol acetic acid) was mixed with the cells suspended in hypotonic solution to reach a final concentration of approximately 20% v/v. Following another centrifugation, the supernatant was discarded and th e cells were treated wi th three changes of fixative. After the third change of fixative, the cell pellet was collected by centrifugation and resuspended in fixative at an approp riate density for slide preparation. The fixed cells were dropped onto clean slides and air-dried before staining. At least two slides were prepared from each culture. Fixed cells not used for slide preparation were discarded after completion of the experimental phase of study.

9.7. Staining At least two slides per culture were washed with 3 changes of purified water (in-house), at approximately 1 minute per change. The washed s lides were stained with 10% v/v Giemsa stain for 15 minutes, washed in one change of purifie d water (prepared in-house) for approximately 1 minute, washed in running tap water for up to 5 minutes, air-dried then mounted with coverslips using synthetic mountant.

9.8. Slide Examination
9.8.1. Mitotic Index (MI) Lymphocyte toxicity is primarily indicated by a decreased MI compared to the concurrent control group. The MI was determined by exam ination of at least 500 cells per culture forTesting Facility Study No.

Page 18 selected treatment groups, i.e., relevant dose levels not showing extreme toxic effects. The relative mitotic index (RMI) for each treatment group was calcu lated as a percentage ratio compared with the appropriate c oncurrent vehicle control group.

9.8.2. Selection of Slides for Detailed Examination Justification for selection of dose levels for examin ation is presented in the results section of this report. Routinely, slides for examination of abe rrations are selected primarily on the basis of the RMI results. For each treatment regime and phase , the highest level selected for examination of aberrations is the highest dose tested in the ca se of test substances showing relatively low toxicity. Otherwise the highest level examined is the lowest concentration which causes a reduction in the RMI to below 50%. On occasion the treatment procedure may have a clear effect on the quality of metaphases or cause an extreme reduction in the absolute numbe r of metaphases, in which case a different maximal dose for examination may be justified. At least two additional lower dose levels of the test substance are assessed. Usually these are th e two adjacent lower dose levels, but this range may be extended at the Study Directorís discreti on if necessary to include at least one level showing little or no toxicity. In addition, one relatively non-t oxic dose level of positive cont rol agent was selected for examination from each of the th ree treatment regimes/phases.

9.8.3. Detailed Examination for Chromosome Aberrations Slides selected for examination were randomized using Microsoft Æ Excel 2000/2003 then encoded to mi nimize potential operator bias. They were examined by light microscopy, and (where practical) a tota l of 200 readable metaphases per expe rimental point were examined for the presence of chromosome abe rrations using oil-immersion optics. Usually, only one slide was examined per culture, the remaining slide(s) being held temporarily in reserve in case of technical problems with the firs t slide, e.g., insufficient metaphases available to complete analysis. Unexamined slides and those not need ed for archiving were discarded after completion of the experimental phase of the study. Readable metaphases are identi fied by the following criteria:

! Chromosome number between 44 and 48 in a single stage of condensation

! Well-spread with minimal overlap of chromosomes and chromosome arms

! Chromatids separate with centromere intact

! Structure of chromosome s clear and well-definedTesting Facility Study No.

Page 19 The International System for Chromosome Ab erration Nomenclature (1995) was followed to designate the observed aberrations . Since the nature of chromosomal and chromatid gaps is uncertain (they may or may not re present true breaks in chromatid structure), these two types of aberration were recorded but were not include d in statistical analys is of aberrations. The incidences of numerica l aberrations, such as pol yploidy and endoreduplication, were recorded. As required by OECD guideline 473, obvious treatment-related increases in the incidence of polyploidy (if present) are described in this report, although the biological relevance of such effects in an in vitro system is unknown. The location (Vernier reading) of observed ab errant metaphases was recorded for potential peer review.

10. EVALUATION AND INTERPRETATION OF RESULTS Results from replicate cultures were combined to facilitate interpretation and maximize the power of statistical analysis. The results obtai ned for each treatment group were compared with the results obtained for the concurrent vehicl e control group from the same treatment regime using Fisherís Exact Test (SAS version 8.1). A positive response is normally indicated by a statistically significan t (dose-related, if applicable) increase in the incide nce of aberrant cells for the tr eatment group compared with the concurrent control group (p ≤ 0.01); individual and/or group m ean values should exceed the laboratory historical control ra nge (99% limit). A negative resu lt is indicated where group mean incidences of aberrant metaphase cells for th e group treated with the test article are not significantly greater than incidences for the concurrent c ontrol group (p > 0.01) and where these values fall within or close to the historical control range. An equivoc al response is obtained when the results do not meet the criteria speci fied for a positive or negative response. For an assay to be considered valid, the vehicle/ negative control results sh ould lie within or close to the historical negative control range, while the positive control should produce a significant increase in the incidence of aberrant cells compared with the concurrent control.

11. QUALITY ASSURANCE The Quality Assurance department of performed and documented inspections and audits of critical phases of the study performed at du ring its conduct and audited the study report. Data audits were conducted according to standard procedures. The Quality Assurance Statement is presented on page 4.Testing Facility Study No.

Page 20 12. PROTOCOL DEVIATIONS Occasional minor deviations fr om the protocol are documented in the raw data and, where appropriate, the text of this report. The inco rrect CAS number was used in the protocol and corrected in this report. No deviation had any impact on the outcome of the study or upon the interpretation of the results.

13. ARCHIVES The following items will be retained in the scientific archives of for a period of approximately 1 year after dispatch of the final report:

! All raw data

! Permanent specimens

! Documents generated at during this study

! The original copy of the protocol (including amendments)

! The final report Subsequent disposition will be doc umented at the Testing Facility.

14. RESULTS
14.1. Dose Selection In the absence of overt toxicity, the highest dose level of the test article selected for detailed analysis was the highest dose leve l tested. In addition, the next two lower dose levels were also subjected to examination.
14.2. Chromosome Aberrations Cultures treated with did not show any statistically significan t increases in the proportion of aberrant metaphase s at any experimental point (see Table 1 for summary and Appendix 2 for detailed results) . In addition, the proportion of aberrant metaphases for all vehicle and test substance groups was within th e laboratory negative hist orical control range (see Figure 1 for negative and posi tive historical control results). The gaseous and conventional positive control agents caused substantial and highly significant increases in the proportion of ab errant metaphases in each phase of the study confirming the sensitivity of the test system and the effectiveness of the S9 mix.Testing Facility Study No.

Page 21 14.3. Incidental Observations No substantial increases in the incidence of ch romatid or chromosome gaps or polyploidy were observed at any experi mental point.

15. CONCLUSION It is concluded that did not show any evidence of genotoxic ity in this in vitro test for induction of chromosome damage when tested in accordance with regulatory guidelines.Testing Facility Study No.

Page 22 Table 1 Results and Statistical Analysis Number of aberrations Incidental observations Ü Treatment Conc. (% v/v) MI RMI (%) Number cells examined % Aberrant b e B E other (g G P C) 4 hours treatment in the absence of S9 (0S9) Vehicle - 9.1 100 200 3.5 4 0 4 0 0 2 0 0 0 Test substance 12.8 9.2 101 200 0.5 1 0 0 0 0 0 0 0 0 25.6 8.2 90 200 1.5 1 0 2 0 0 3 0 0 0 50.0 8.6 95 200 3.5 4 0 3 0 0 0 0 1 0 ETOX 1.0 6.7 73 200 12.0** 18 3 5 0 0 0 0 0 0 MMC 0.10 a 8.8 97 200 16.0** 23 8 7 0 0 1 0 0 0 4 hours treatment in the presence of S9 (+S9) Vehicle - 7.2 100 200 0.0 0 0 0 0 0 4 0 1 1 Test substance 12.8 8.4 116 200 1.5 2 0 1 0 0 2 0 0 0 25.6 8.1 112 200 2.0 4 0 1 0 0 0 0 1 0 50.0 8.7 121 200 0.5 1 0 0 0 0 3 0 0 0 ETOX 0.5 6.5 91 200 8.5** 12 2 6 0 0 0 1 1 0

CP 8.0 a 4.1 57 200 27.5** 39 4 33 0 0 9 0 0 0 21 hours treatment in the absence of S9 (0S9) Vehicle - 7.9 100 200 0.5 1 0 0 0 0 3 0 1 0 Test substance 12.8 6.7 84 200 1.5 0 0 3 0 0 1 0 0 0 25.6 5.7 73 200 3.0 2 0 5 0 0 2 0 0 1 50.0 6.7 85 200 2.0 4 0 0 0 0 3 1 0 0 ETOX 0.5 4.4 56 200 9.5** 16 2 5 0 0 2 0 0 1 MMC 0.05 a 7.5 94 200 14.5** 12 5 16 0 0 0 1 0 0 MI, RMI Mitotic Index, Relative Mitotic Index (vehicle = 100%) a Standard positive controls dosed as solutions; in units of μg/mL b, e, g Chromatid break, exchange, gap B, E, G Chromosome break, exchange, gap other Includes pulverized chromosomes and cells with > 8 aberrations P Polyploidy and endoreduplication C Centromeric disruption Ü g, G, P and C are excluded from the calculation of % aberrant cells Results of statistical analysis using one-tailed Fisherís exact test * p ≤ 0.01 (significant) ** p ≤ 0.001 (highly significant) Otherwise, p > 0.01 (not significant)Testing Facility Study No.

Page 23 Figure 1 Historical Control Values

305

132

64 26 18 8 4 1 000 0 50 100 150 200 250 300 350

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 >5 Mean % aberrant cellsFrequency

The laborator y historical mean incidence of aber rant metaphase cells for negative/vehicle control cultures for the human lymphocyte chromosome aberration test is 0.43% (SD 0.63) for 558 treatments. These QA audited results were collected from GLP compliant studies performed from 05 February 2003 to 19 February 2009. The historical positive control values (for QA- audited and GLP compliant studies) are listed below: Mitomycin C (4 hour 0S9): mean 10.3%, SD 5.0, 144 treatments Mitomycin C (21 hour 0S9): mean 11.8%, SD 4.7, 149 treatments Cyclophosphamide (4 hour +S9): mean 21.2%, SD 7.7, 148 treatmentsTesting Facility Study No.

Page 24 Appendix 1 Certific ate of AnalysisTesting Facility Study No.

Page 25Testing Facility Study No.

Page 26 Appendix 2 Results for Individual CulturesTesting Facility Study No.

Page 27 Appendix 2 Results for Individual Cultures

Number of aberrations Incidental observations Treatment Conc. (% v/v) MI Number cells examined Number aberrant cells b e B E other (g G P C) 4 hours treatment in the absence of S9 (0S9) Vehicle - 7.7 100 5 3 0 3 0 0 1 0 0 0 10.5 100 2 1 0 1 0 0 1 0 0 0 Test substance 12.8 7.9 100 0 0 0 0 0 0 0 0 0 0 10.6 100 1 1 0 0 0 0 0 0 0 0 25.6 8.9 100 3 1 0 2 0 0 1 0 0 0 7.5 100 0 0 0 0 0 0 2 0 0 0 50.0 7.8 100 5 3 0 2 0 0 0 0 1 0 9.4 100 2 1 0 1 0 0 0 0 0 0 ETOX 1.0 5.4 100 13 9 3 3 0 0 0 0 0 0 8.0 100 11 9 0 2 0 0 0 0 0 0 MMC 0.10 a 10.9 100 19 12 6 2 0 0 0 0 0 0 6.7 100 13 11 2 5 0 0 1 0 0 0 4 hours treatment in the presence of S9 (+S9) Vehicle - 6.2 100 0 0 0 0 0 0 2 0 1 0 8.2 100 0 0 0 0 0 0 2 0 0 1 Test substance 12.8 7.7 100 1 0 0 1 0 0 0 0 0 0 9.0 100 2 2 0 0 0 0 2 0 0 0 25.6 7.5 100 3 3 0 1 0 0 0 0 0 0 8.7 100 1 1 0 0 0 0 0 0 1 0 50.0 10.2 100 0 0 0 0 0 0 1 0 0 0 7.1 100 1 1 0 0 0 0 2 0 0 0 ETOX 0.5 7.3 100 9 8 2 2 0 0 0 1 0 0 5.7 100 8 4 0 4 0 0 0 0 1 0 CP 8.0 a 4.2 100 26 16 3 18 0 0 2 0 0 0 4.1 100 29 23 1 15 0 0 7 0 0 0 21 hours treatment in the absence of S9 (0S9) Vehicle - 7.4 100 1 1 0 0 0 0 2 0 0 0 8.4 100 0 0 0 0 0 0 1 0 1 0 Test substance 12.8 7.7 100 2 0 0 2 0 0 0 0 0 0 5.6 100 1 0 0 1 0 0 1 0 0 0 25.6 6.0 100 4 2 0 3 0 0 0 0 0 1 5.5 100 2 0 0 2 0 0 2 0 0 0 50.0 5.6 100 2 2 0 0 0 0 2 1 0 0 7.8 100 2 2 0 0 0 0 1 0 0 0 ETOX 0.5 3.5 100 9 8 2 3 0 0 1 0 0 0 5.3 100 10 8 0 2 0 0 1 0 0 1 MMC 0.05 a 8.0 100 16 8 3 7 0 0 0 0 0 0 6.9 100 13 4 2 9 0 0 0 1 0 0 For explanatory footnotes, please see Table 1.Testing Facility Study No.

Page 28 Appendix 3 Protocol and AmendmentsTesting Facility St udy No.

Page 29Testing Facility St udy No.

Page 30Page 3 Testing Facility Study No.

1. STUDY SPECIFIC DETAILS Purpose To evaluate the genotoxicity of th e test substance using an in vitro mammalian chrom osome aberration test. Sponsor's Representative

Testing Facility

Compliance Statement This study will be conducted in accordance with the OECD Principles of Good Laboratory Practice (revised 1997, published 1998) and EPA GLP Standard 40 CFR Part 792 (TSCA) with the following exceptions:

● Concentration, stability and homoge neity of test atmosphere will not be determined in this study.

● Test substance characterization was not conducted in compliance with GLP. Characterization of the test substance was conducted by the Sponsor’s laboratory accordin g to established SOPs, controls and approved test methodol ogies to ensure integrity and validity of the results generated. The procedures described in this protocol comply with the recommendations of the ICH S2A (F ederal Register. Adopted 1995; 61:18197-18202), the OECD guideli ne 473 for the testing of chemicals (1997), the US EPA health effects test guidelines (1998, Testing Facility Study No.

Page 31Page 4 Testing Facility Study No. OPPTS 870.5375) and the US FDA Redbook 2000 (Short-Term Tests for Genetic Toxicity, July 7, 2000). Archiving Period Approximately 1 year at the Testing Facility.
Test Substance Identity CAS number Chemical name Molecular weight Action (intended use) Industrial chemical Lot number
Description Colorless g as under norm al room conditions, boiling point 7.5°C Storage conditions Kept in a pressurized cylinder in a ve ntilated area at ambient room temperature Supplier Sponsor Purity/Correction Assume d to be 100%; all concentra tions to be expressed in terms of material as is.

Vehicle/Diluent Ambient laboratory air. Test Atmosphere Preparation Appropriate mixture prepared in s itu immediately at the start of exposure Stability of Test Substance Atmosphere Will not be determined in this study.

Formulation Analysis for Achieved Concentration Will not be determined in this study Testing Facility Study No.

Page 32Page 5 Testing Facility Study No. Study Design
Culture numbers Dose Number Material Exposure conc. % v/v 4 Hours (0S9) 4 Hours (+S9) 21 Hours (0S9) 0 Vehicle control - a01, a02 b01, b02 c01, c02 1 Test substance 0.1 a 11, a12 b11, b12 c11, c12 2 0.2 a21, a22 b21, b22 c21, c22 3 0.4 a31, a32 b31, b32 c31, c32 4 0.8 a41, a42 b41, b42 c41, c42 5 1.6 a51, a52 b51, b52 c51, c52 6 3.2 a61, a62 b61, b62 c61, c62 7 6.4 a71, a72 b71, b72 c71, c72 8 12.8 a81, a82 b81, b82 c81, c82 9 25.6 a91, a92 b91, b92 c91, c92 10 50.0 a101, a102 b101, b102 c101, c102 1 ETOX 0.5 a111, a112 b111, b112 c111, c112 2 1.0 a113, a114 b113, b114 c113, c114 3 2.0 a115, a116 b115, b116 c115, c116 1 Mitomycin C a 0.05 a121, a122 - c121, c122 2 0.10 a123, a124 - c123, c124 3 0.20 a125, a126 - c125, c126 1 Cyclophosphamide a 8.0 - b121, b122 - 2 12 - b123, b124 - 3 16 - b125 , b1 26 - Where high level = maximum practical without resulting in anoxia. The dose interval may be modified slightly at the time of the experiment for practical reasons. The 21 hour incubation ('confirmatory phase') is required by regulatory authorities where the initial phase does not show any indication of genotoxicity for the test substance; however, it is routinely performed at the same time as the initial phase to minimize potential delays in performance of the study.
a Standard positive controls dosed as solutions; in units of μg/mL

Modifications and Notes Gas testing procedures as per A ppendix 6 of Testing Facility SOP

2. REASON FOR CHOICE OF TE ST SYSTEM Primary cultures of human peripheral lymphoc ytes are preferred becau se of their stable chromosome number and structure (karyotype) compared with cell lin es, and their low and stable background rate of aberrations. Testing Facility Study No.

Page 33Page 6 Testing Facility Study No.
3. REASON FOR DOSE LEVEL S ELECTION The highest dose chosen is approximately th e maxim um without causing anoxia. The dose interval of 2 is the minimum standard recommended by the OECD guideline.

4. PARTICIPATING PERSONNEL Study Director

Participatin g Scientist(s)

5. SCHEDULE

Start 11 March 2010 Draft repor t (non-audited) 29 April 2010 Suggested date for client comments Within 3 weeks of receipt of draft report (see below) Final report See below In order to ensure compliance with GLP, if no reque sted revisions or instru ctions to finalize have been communicated by the Sponsor six months afte r issuance of the draf t report, the report will be issued as the signed final version follo wing QA audit and review. Otherwise, report finalization norma lly occurs about 4 weeks fo llowing receipt of client comments and any contributing reports. Testing Facility Study No.

Page 34Page 7 Testing Facility Study No.
6. COMPUTER SYS TEMS 3⁄4 Statistical analysis involves l ook-up tables generated using valid ated statistical methods in SAS version 8.1.
3⁄4 Arithm etic calculations of mean values etc fo r presentation in re ports are made using Microsoft Excel 2000/2003.

7. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS
7.1. Documentation Full details of the vehicle, test and positive c ontrol substances, includ ing supplier, appearance, lot number, storage, expiry date, formulation met hods and concentrations (if applicable), will be recorded in the raw data; appropriate detail s will be presented in the final report.

7.2. Vehicle Any solubility or compatibility testing for vehicl e selection in this test system is normally performed in accordan ce with Testing Facility SOPs but, since it is not protocol-driven, is necessarily non-GLP. Records (o r true copies) of the solubility testing, if done, will be filed alongside the GLP study raw data.

7.3. Test Substance and Sponsor's Responsibilities The batch/lot number, concentra tion, purity, expiry date, descri ption, handling precautions and supplier will be provided by the Sponsor. The appr opriate details will be included in the final report. The test substance should be characterized by th e Sponsor by performing analyses according to GLP regulations – refer to STUDY SPECIFIC DETA ILS for any exceptions. All data relevant to such analyses will b e reviewed, audited and retained by the Sponsor who will provide a test substance Certificate of Analysis for inclusion in the study report. The Sponsor’s approval of the pr otocol indicates that appropri ate documentation of the me thod of synthesis, fabrication or de rivation of the test and any non- commercial control substance(s) supplied by the sponsor is on file and that it is available to the appropriate regulatory agencies should it be requested. Testing Facility Study No.

Page 35Page 8 Testing Facility Study No. Unless specifically requested by the Sponsor, all test substanc e remaining at study completion will be retu rned to the Sponsor (storage conditio ns, contact, address, fax number and telephone to be provided). Alternativel y, the test substance can be reta ined at for subsequent studies. The final disposition of the test subs tance at will be documented but not reported unless specifically requested by the Sponsor.

7.4. Routine Positive Controls Mitomycin C and Cyclophosphamide will be used as positive control agents. The positive control formulations will not be subjected to analysis for safety reasons and because the biological response of the test syst em is considered to be the best measure of the appropriateness of the formulations.

7.5. Gaseous Positive Controls The gaseous positive control will be mixed in a ppropriate proportions with am bient laboratory air in situ in the culture tubes. Identity: Ethylene oxide (ETOX) Vehicle: Ambient laboratory air Concentration: 0.5 , 1.0 and 2.0% v/v

8. EXPERIMENTAL PROCEDURES
8.1. Test System
8.1.1. Blood Sampling A peripheral blood sample will be taken from healthy, non-smoking, ma le donor(s) into blood collecting tu bes containing sodium heparin and will be held at room temperature for up to 2 hours prior to culture initiation.

8.1.2. Culture Medium Complete RPMI 1640 medium will be prepared by supplementing RPMI 1 640 medium with the following filter-sterilized component s: 10% (v/v) fetal calf serum, 50 μg gentamycin per mL and 4 units heparin per mL. Testing Facility Study No.

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8.1.3. Lymphocyte Culture Phytohemagglutinin will be added to whole blood mixed with m edium to stimulate lymphocyte division. Aliquots of 5 mL cell su spension will be incubated in fl at-sided culture tubes (Nunc) at approximately 37°C in a humidifi ed atmosphere containing 5% CO 2 .
8.2. S9 Mix The S9 mix, used as a model of intact mammalian metabolism, will be prepared on the day of use and will contain 10% v/v S9 fraction (phenobarbital/5, 6-benzoflavone induced male Sprague-Dawley rat liver fraction supplied by Moltox TM ) and the following sterile cofactors: 8 mM MgCl 2 , 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP . The S9 mix will be kept in a refrigerator set to maintain 4°C or on ice until required. A copy of the manu facturer’s quality contro l certificate(s) for the S9 fraction will be retained as raw data.

8.3. Treatment Treatments will be performed approximately 48 hours after culture ini tiation. For the S9 cultures, 1 mL of medium will be removed and replaced with 1 mL S9 mix. The cell suspensions to be treated with gaseous test substances will be transferred to gas-tight tubes (24 mL glass anaerobic culture tubes supplied by Be llco Glass) and each tube will be sealed with a gas impermeable, butyl rubber septum (injectab le) stopper and an aluminum seal. Using a gas-tight syringe and needle, the appropriate vol ume of air will be removed from each treatment tube, then the appropriate volume of treatment ga s will be added. For the cell suspensions to be treated with the routine positive controls, the a ppropriate volume of liquid test substance will be added to the cultures in the original 5 mL flat-sid ed tubes. All the cultures will be incubated at
ca. 37°C in a humidified atmosphere containing 5% v/v CO 2 . Several concentrations of the test substance will be tested using all treatment regimes (4 hour treatment in the absence and presence of S9 mix and a 21 hour treatment in the absence of S9). The 21 hour incubation (‘confirmatory phase’) is required by regulatory aut horities where the initial phase does not show any indication of genotoxicity for the test substa nce; however, it is rou tinely performed at the same time as the initial phase to minimize pot ential delays in perf ormance of the study. Duplicate cultures will be treate d at each experimental point.

8.4. Medium Change (4-hour Treatment only) After the 4-hour treatment, the 4-hour gas-treate d cultures will be opened in a lam inar flow hood to allow the gas to dis sipate and the cells will be transferred to flat-sided culture tubes (Nunc). All 4-hour treatment cultures will then be centrifuged at 1000 rpm for 5 minutes. The supernatant will be discarded and replaced with fresh complete medium. The cultures will be Testing Facility Study No.

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returned to the incubator for another 17 hours of incubation before metaphase harvesting. For 21-hour treated cultures (i.e. cu ltures in the conf irmatory phase), no replacement of medium will be performed.

8.5. Harvesting, Metaphase Preparation and Staining After colcemid addition (0.1 μg/mL), the culture s will be incubated for approximately 2 hours prior to harv esting. After 2 h, th e gas-treated cultures from the 21h0S9 regime will be opened in a laminar flow hood to allow the gas to dissipate and the cells will be tran sferred to flat-sided culture tubes (Nunc). Cultures will be harv ested by centrifugation. The cell pellet will be resuspended in hypotonic solution (0.075M KCl) and incubated at ca. 37°C. Fixative will be mixed with the suspended cells and following an other centrifugation, the cells will be treated with 3 changes of fixative. After the third change of fixative, the cell pellet will be resuspended in fixative at an appropriate density for slide preparation. The fixed cells will be dropped onto clean slides and air-dried before staining. At least two slides will be prepared from each culture. Fixe d cells not used for slide preparation will be discarded after completion of the experimental ph ase of study. Slides will be washed, stained with Giemsa, rinsed, air-dried then mounted with coverslips.

9. SLIDE EXAMINATION
9.1. Mitotic Index (MI) Lymphocyte toxicity is norm ally indicated by a decreased MI compared to the concurrent control group. The MI will be d etermined by examination of at least 500 cells (if available) from selected treatment groups, i.e . relevant dose levels not showin g extreme toxic effects. The relative mitotic index (RMI) for each treated group will be calculated as a percentage compared with the concurrent vehicle control group.

9.2. Selection of Slides for Detailed Examination Justific ation for selection of dose levels for examination will be presented in the results section of the report. Routinely, slides for examination of aberration s are selected primarily on the basis of the RMI results. For each treatment regime an d phase, the highest dose le vel tested is selected for examination of aberrations unl ess there is toxicity (in which case, the highest level examined is the lowest concentration which causes a reduction in the RMI to below 50%). On occasion the treatment procedure may have a clear effect on the quality of metaphases or cause an ex treme reduction in the absolute numbe r of metaphases, in which case a different Testing Facility Study No.

Page 38Page 11 Testing Facility Study No. maximal dose for examination may be justified. At least two additional lower dose levels of the test substanc e will be assessed. Usually these ar e the two adjacent lower dose levels, but this range may be extended at the Study Director's disc retion if necessary to include a least one level showing little or no toxicity. In addition, one relatively non-t oxic dose level of positive cont ro l agent will be selected for examination from each of the th ree treatment regimes/phases.

9.3. Detailed Examination for Chromosome Aberr ations Slides selected for examination will be randomized using Microsoft ® Excel 2000/2003 then encoded to minimize potential operator bias. They will be examined by light microscopy, and (where practical) a total of 200 readable metaphase s per experimental point will be examined for the presence of chromosome abe rrations using oil-immersion optics. Slides from cultures not subjected to analysis for chromosome aberrations or needed for archiving, will be discarded after completion of the experimental phase of study. Readable metaphases are identi fied by the following criteria:

■ chromosome number between 44 and 48 in a single stage of condensation
■ well-spread with mini mal overlap of chromosomes and chromosome arms
■ chromatids separate with centromere intact
■ structure of chromosome s clear and well-defined The International System for Chromosome Aberration Nomenclature (1995) will be followed to designate the observed aberrations . Since the nature of chromosomal and chrom atid gaps is uncertain (they may or may not re present true breaks in chromatid structure), these two types of aberration will be recorded but will not be included in statistical analysis of aberrations. The incidences of numerical types of aberration, su ch as polyploidy and endoreduplication, will be recorded. As required by OECD guideline 473, obvious treatment-relate d increases in the incidence of polyploidy (if present) will be desc ribed in the report, although the biological relevance of such effects in an in vitro test is unknown. The location (Vernier reading) of observed aberrant metaphases will be recorded for potential peer review.

10. EVALUAT ION AND I NTERPRETATION OF RESULTS Results from replicate cultures will be combined to facilitate interpretation and maximize the power of statistical analysis. The results obt ained for each treatm ent group will be compared Testing Facility Study No.

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with the results obtained for the concurrent vehicle control group from the same treatment regime using Fisher’s Exact Test (SAS version 8.1). A positive response is normally indicated by a statistically significan t (dose-related, if applicable) increas e in the incide nce of aberrant cells for the tr eatment group compared with the concurrent control group (p ≤ 0.01); individual and/or group m ean values should exceed the laboratory historical control ra nge (99% limit). A negative resu lt is indicated where group mean incidences of aberrant metaphase cells for th e group treated with the test substance are not significantly greater than incidences for the concurrent c ontrol group (p > 0.01) and where these values fall within or close to the historical control range. An equivoc al response is obtained when the results do not meet the criteria specified for a positive or negative response. Additional slide reading or experimental work may be required by the Study Director to clarify results. For an assay to be considered valid, the vehicle/ negative control results sh ould lie within or close to the histo rical control range, while the positiv e control should produce a significant increase in the incidence of aberrant cells comp ared with the concurrent control.

11. QUALITY ASSURANCE The Quality Assurance department of will undertake and document inspections and audits of critical phases of the study perfor med at during its conduct and audit the study report. Data audits will be conducted according to standard procedures. Authorized designates of the Sponsor may insp ect their study during regular working hours for quality assurance purposes.

12. ARCHIVES The fo llowing items will be retained in the scientific archives of for the period described in the STUDY SPECIFI C DETAILS section after disp atch of the final report: All raw data Permanent specimens Documents generated at during this study The original copy of the protocol (including amendments, if any) The final report Testing Facility Study No.

Page 40Page 13 Testing Facility Study No. Subsequent disposition will be docum ented at the Testing Facility.

13. COMMUNICATION Untoward changes or events occurring during th e study will be communicat ed to the Sponsor by telephon e, FAX or em ail. Agreed upon changes in study conduct or design will be documented in protocol amendments. Hard and/or electronic copies of each protocol am endment will be sent to the Sponsor who will return formal approval of the document to

14. REPORTS A com plete detailed non-audited draft report will be submitted to the Sponsor electronically for review. Any revised draf t reports will be subm itted to the Sponsor electronically for further review. Subsequent to the in clusion of any modifications or corrections (agreed upon by the Sponsor and a submission ready version (linked, searchable PDF) of the final report will be provided electronically. If no requested revisions or instructions to finalize have been communicated by the Sponsor six months after issu ance of the draft report , the draft report will be issued as a signed final report. Testing Facility Study No.

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Suggested date for client comments Within 2 weeks of receipt of draft report (see below) Final report June 2010, see below In order to ensure compliance with GLP, if no reque sted revisions or instru ctions to finalize have been communicated by the Sponsor six months afte r issuance of the draf t report, the report will be issued as the signed final version follo wing QA audit and review. Otherwise, report finalization norma lly occurs about 4 weeks fo llowing receipt of client comments and any contributing reports. Justification: To include the experimental co mple tion date and specify final report issuance timeline as requested by the Sponsor. Testing Facility Study No.

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