FINAL REPORT

Unpublished Work

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



### Bacterial Reverse Mutation A ssay Using Gas-phase Exposure

SPONSOR:

STUDY COMPLETED ON 03 June 2010

Page 1 of 50Testing Facility Study No.

Page 5 TABLE OF CONTENTS

1. COMPLIANCE STATEMENT ...........................................................................................2 2. QUALITY ASSURANCE STATEMENT...........................................................................3 3. CERTIFICATION STATEMENT ......................................................................................3 LIST OF TABLES .........................................................................................................................7 LIST OF FIGURES .......................................................................................................................8 LIST OF APPENDICES ...............................................................................................................9 4. STUDY INFORMATION...................................................................................................10 5. SUMMARY INFORMATION...........................................................................................11 6. INTRODUCTION ...............................................................................................................12 7. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS ..................................12 7.1. Control Atmosphere...................................................................................................12 7.2. Test Substance ............................................................................................................12 7.3. Positive Controls.........................................................................................................12 7.3.1. In the Absence of S9 Mix ............................................................................12 7.3.2. In the Presence of S9 Mix ...........................................................................13 8. EXPERIMENTAL PROCEDURES..................................................................................13 8.1. Test System .................................................................................................................13 8.2. S9 Mix..........................................................................................................................14 8.3. Sterility Controls ........................................................................................................14 8.4. Atmosphere Concentration and Stability.................................................................14 8.5. Test Method and Test Atmosphere Generation ......................................................14 9. EVALUATION AND INTERPRETATION OF RESULTS ...........................................16 10. QUALITY ASSURANCE...................................................................................................18 11. PROTOCOL DEVIATIONS..............................................................................................18 12. ARCHIVES..........................................................................................................................18 13. RESULTS........................................................................................................................ .....18Testing Facility Study No.

Page 6 13.1. Bacterial Mutation Test .............................................................................................18 14. CONCLUSION....................................................................................................................19Testing Facility Study No.

Page 7 LIST OF TABLES

Table 1 - Initial Test in the Absence of S9 Mix.................................................. 20 Table 2 - Initial Test in the Presence of S9 Mix ................................................. 21 Table 3 Positive Controls for the Initial Test..................................................................... 22 Table 4 - Confirmatory Test in the Absence of S9 Mix ..................................... 23 Table 5 - Confirmatory Test in the Presence of S9 Mix..................................... 24 Table 6 Positive Controls for the Confirmatory Test......................................................... 25Testing Facility Study No.

Page 8 LIST OF FIGURES Figure 1 TA1535 Revertant Colony Count Historical Results............................................ 26 Figure 2 TA1537 Revertant Colony Count Historical Results............................................ 26 Figure 3 TA98 Revertant Colony C ount Historical Results................................................ 27 Figure 4 TA100 Revertant Colony Count Historical Results.............................................. 27 Figure 5 WP2 uvrA Revertant Colony Count Historical Results ........................................ 28Testing Facility Study No.

Page 9 LIST OF APPENDICES Appendix 1 Certificate of Analysis .......................................................................................... 29 Appendix 2 Historical Positive Control Results....................................................................... 31 Appendix 3 Protocol and Amendments.................................................................................... 33Testing Facility Study No.

Page 10 4. STUDY INFORMATION Substance Tested : ï CAS Number) Number : 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 : 09 March2010/(see report cover page) Experimental Start/Termination : 12 March 2010/30 March 2010Testing Facility Study No.

Page 11 5. SUMMARY INFORMATION The purpose of this study was to evaluate the ge notoxicity of using the bacterial reverse mutation test. Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia coli strain WP2 uvrA were treated with the gaseous test substan ce at a range of concen trations separated by a half-log 10 dose interval up to 100%, i.e., the maxi mu m practical level) in the presence and absence of a supplemented liver fraction (S9 mix) using the plate incor poration version of the bacterial mutation test. Since no evidence of genot oxicity and only slight toxicity was seen in this initial test, the study was repeated using a narrower (approximately 2-fold) dose interval to confirm the results. Bacteria were incubated with standard positive control agents, and the response of the various bacterial strains to these agents confirmed the se nsitivity of the test system and the activity of the S9 mix. No substantial increases in the revertant colony counts were obtained with any strain following exposure to the test substance in either the ini tial or confirmatory assa ys in the absence or presence of S9 mix. It is concluded that did not show any evidence of genotoxic activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.Testing Facility Study No.

Page 12 6. INTRODUCTION The bacterial mutation test detects point mutatio ns, which are the cause of many human genetic diseases and can play a role in tumor initiation and development.
7. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS 7.1. Control Atmosphere Ambient laboratory air was used to treat negative/vehicle control plates and as the diluent for the test substance.
7.2. Test Substance Identity CAS number Chemical name Molecular weight Lot number Expiry date 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 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).
7.3. Positive Controls 7.3.1. In the Absence of S9 Mix Identity Sodium azide (NaAz) CAS number 26628-22-8 Vehicle Sterile water for irrigation USP Supplier Sigma-AldrichTesting Facility Study No.

Page 13 Identity 9-Aminoacridine (9AC) CAS number 52417-22-8 Vehicle Sterile water for irrigation USP Supplier Sigma-Aldrich Identity 2-Nitrofluorene (2NF) CAS number 607-57-8 Vehicle DMSO Supplier Sigma-Aldrich Identity 4-Nitroquin oline N-oxide (NQO) CAS number 56-57-5 Vehicle DMSO Supplier Sigma-Aldrich
7.3.2. In the Presence of S9 Mix Identity 2-Aminoanthracene (2AA) CAS number 613-13-8 Vehicle DMSO Supplier Sigma-Aldrich Identity Benzo[a]pyrene (BaP) CAS number 50-32-8 Vehicle DMSO Supplier Supelco Full details of positive controls including expiry da te, lot number, appearance, storage, copies of certificates of analysis and formul ation are retained as raw data.
8. EXPERIMENTAL PROCEDURES 8.1. Test System The bacteria were originally supplied by Moltox ô, NC, USA. Each batch of frozen bacteria was tested for appropriate phenotyp e characteristics and spontaneous reversion rates; response to diagnostic mutagens is also routinely assessed. The following bacterial strains were employed:Testing Facility Study No.

Page 14 S. typhimurium TA1535 his G46 rfa ∆ uvrB S. typhimurium TA1537 his C3076 rfa ∆ uvrB S. typhimurium TA98 his D3052 rfa ∆ uvrB pKM101 S. typhimurium TA100 his G46 rfa ∆ uvrB pKM101 E. coli WP2 trp uvrA Fresh bacterial cultures were prepared so that th ey were in the late-log phase of growth at the time of use. The density of th e cultures was confirmed to be ≥ 1000 × 10 6 bacteria/mL using a bacterial counting chamber before the cultures were used in the test.
8.2. 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 Moltox, stored in a freezer set to maintain -80°C, thawed before use) and the followi ng 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 stored at in a refrigerator set to mainta in 4°C or on ice until required. A copy of the manufacturerís quality control certificate(s) for the S9 fraction is retained as raw data.
8.3. Sterility Controls Sterility of appropriate components used in the test including buffer, S9 mix, positive controls (high dose where more than one dose is tested ) was confirmed on the day of the test using appropriate preparations without bacteria (detailed results not reported here). The spontaneous mutation rates of the bacterial strains were assessed using concurrent control samples in which the bacteria were expos ed to the control atmosphere (air). 8.4. Atmosphere Concentration and Stability Chemical analysis of test atmosphere for test substance content and stability was not performed 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.
8.5. Test Method and Test Atmosphere Generation The study employed the standard plate incorporati on method. In the initial test, a 0.5 mL aliquot of S9 mix (+S9) or phosphate buffer 0.2M pH 7.4 (0S9) was combined with 0.1 mL bacterialTesting Facility Study No.

Page 15 culture in a sterile container. Where appropriate, a standard volume (0.1 mL) of the test positive control was added. Approximately 2 mL of mo lten top agar supplemented with 0.05 mM biotin and minimal (0.05 mM) histidine and minima l (0.05 mM) tryptophan was added immediately afterward. The solution was mixed and overl aid onto a minimal gluc ose plate (1.5% agar, Vogel-Bonner medium E, 2% glucose). After the ove rlay solidified, the plat es were inverted and incubated at ca. 37°C for 48 to 72 h. The sterile containers and each plat e were labeled with: study number, a unique number that identifies the contents by reference to the experimental design held with the raw data, a nd the day code. Afte r the overlay solidified, the plates were inverted, stacked and placed in Tedlar bags that had been slit on one side (one bag per treatment). The bags were sealed with adhesi ve tape and the excess ai r inside the bags was evacuated - note that this initial evacuation wa s omitted from the protocol in error, but was correctly performed as per SOP and pro cedural records in the study raw data. Appropriate volumes of ambien t laboratory air and stock te st gas were added via the septum/valve into each bag to achieve the fina l concentrations indica ted in the Study design. For the highest dose of the test gas, the gas wa s added to the bag, then purged prior to the addition of a second volume of gas. The plates in the bags were transferred to incubators at ca. 37°C for 48 to 72 h. The sterility plates for the buffer and S9 mix as well as the plates treated with the routine liquid positive controls were not placed in Tedlar bags. They were inverted and incubated at ca. 37°C for the same duration as the test atmosphere-treated pl ates. After the treatment period, the bags were opened in a fume hood or extracted laminar flow cab inet and the plates allowed to equilibrate for ca. 20 mins before counting colonies. The initial test used a range of dose levels separated by a half-log 10 dose interval up to the ma ximum practical limit (100% Based on th e results of the initial test, a confirmatory test was performed using the same methodology and the same upper exposure level but, in this case, a narrow (approximately 2-fold) c oncentration interv al was employed.Testing Facility Study No.

Page 16 Text Table 1 Study Design for Initial and Confirmatory Tests Number of replicates Dose number Material Exposure conc. % v/v 0S9 +S9 Number of strains Initial test 0 Vehicle

- 3 3 5 1 Test substance 0.032 3 3 5 2 0.100 3 3 5 3 0.316 3 3 5 4 1.000 3 3 5 5 3.16 3 3 5 6 10.0 3 3 5 7 31.6 3 3 5 8 ~100 a 3 3 5 Positive con trol á 3 3 5 Confirmatory test 0 Vehicle

- 3 3 5 1 Test substance 3.2 3 3 5 2 6.4 3 3 5 3 12.8 3 3 5 4 25.6 3 3 5 5 51.2 3 3 5 6 ~100 3 3 5 Positive control á 3 3 5 a The maximum practical dose (nominally 100%) was achiev ed by removing as much air as possible from the bag using a vacuum pump, gassing with test substance, purging the atmosphere then re-gassing. á Depends on the test organism and positive control agent. 9. EVALUATION AND INTERPRETATION OF RESULTS After the incubation/equilibration period, the plates were examined visually and, if necessary, with the aid of an inverted microscope. Plat es were evaluated for the quality of the background lawn and the number of revertant colonies. Re vertant colony counts were routinely collected and saved directly into an electronic database using a ProtoCOL automated colony counter (version 3.06 supplied by Synbiosis). The electr onic raw data were transferred to CD-ROM and held with the rest of th e study data. Colony numbers were enumer ated visually if precipitation or other artifacts interfered with the colony counter or at the di scretion of the Study Director. Visual counts and the presence of visible precipitate, if any, were manually recorded in the raw data.Testing Facility Study No.

Page 17 If available, the plates from at least five non-toxic dose levels of the test substance were assessed in each experiment, i.e., the five highest levels be low the toxic level. Toxic effects of the test substance are normally indicated by the partial or complete absence of a background lawn (colony counts, if any, are not reported) or a su bstantial dose-related re duction in revertant colony counts compared with lower dose levels and concurrent vehicle controls (i.e., fold response < 0.6) taking into account the laborator y historical control range. Where five (relatively non-toxic) dose leve ls were analyzed, the remaining plates from lower dose levels may not have been evaluated, and are not reported. Means and standard deviations for appropriate dose levels were calc ulated using Microsoft Æ Excel. The mean number of reve rtant colonies for all treatment groups was compared with those obtained for the concurrent vehi cle control group. The mutagenic activity of the test substance was assessed by applying the following criteria: 1. The results are considered positive (i.e. indicative of mutagenic potential) if both the following criteria are met.

! The results for the test substance show a substantial increase in revertant colony counts, i.e. response ≥ 2 times the concurrent vehi cle control values for TA98, TA1535, TA1537 and WP2 uvrA , or ≥ 1.5 times for TA100, with mean value(s) outside the laboratory histor ical control range (beyond the 98% tolerance limit). Otherwise results are considered negative.

! The above increase must be dose related an d/or reproducible i.e. increases must be obtained at more than one experiment al point (more than one strain or dose level, more than one occasion or with different methodologies). 2 If the second criterion is not met, further te sting might be appropriate to clarify equivocal results using an appropriately modified study design, e.g., a narrower dose interval with the appropriate strain. In such cases, if no substantial increase is obtained in the confirmatory test, the results would be considered negative. For an assay to be considered valid, the mean re vertant colony counts of the vehicle controls for each strain should lie close to or within the current historical control range of the laboratory. All positive control s ubstances (with S9 mix where requi red) should produce increases in revertant colony numbers to at least twice the concurrent vehi cle control levels with the appropriate bacterial strain (1.5× for strain TA100). Any values that do not comply with these criteria are discussed in the Re sults section of this report.Testing Facility Study No.

Page 18 10. QUALITY ASSURANCE The Quality Assurance department of performed and documented inspections and audits of critical phases of the study performed at during its conduct and audited the study report. Data audits were conducted according to standard procedures. The Quality Assurance Statement is presented on page 3.
11. PROTOCOL DEVIATIONS Occasional minor deviations from the protocol, if any, are documented in the raw data and, where appropriate, the text of this report. No deviation had any impact on the outcome of the study or upon the interpre tation of the results.
12. 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

! Documents generated at during this study

! The original copy of the protocol (including amendments)

! The final report Subsequent storage details will be documented in the raw data.
13. RESULTS 13.1. Bacterial Mutation Test Occasional reductions in the revertant colony count were obtained with some bacterial strains at the highest concentration of indicating th at the substance was somewhat toxic to the bacteria and confirming exposure. Revertant col ony counts were recorded and reported here for at least the five highest n on-toxic concentrations of the test substance; i.e. at least the five highest non-toxic concentrations. The results are pr esented in Table 1, Ta ble 2, Table 3, Table 4, Table 5 and Table 6; footnotes ar e explained in Table 1. The mean and standard deviation values quoted have been rounded to the nearest whole number. The absence of colonies on sterility check plates confirmed absence of microbial contamination (results not shown). The mean re vertant colony counts for the negativ e controls were close to or within the laboratory historic al control range (see Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5 for individual colony c ount historical results). Appr opriate positive control compounds (with S9 mix where required) induc ed increases in revertant colony numbers to at least twice the concurrent vehicle control levels with the appr opriate bacterial strain (1.5× for strain TA100),Testing Facility Study No.

Page 19 confirming sensitivity of the test system and activity of the S9 mix (see also Appendix 2 for positive control historical results). No substantial increases in revertant colony numbers were obtained with any of the tester strains following exposure to at any concentration in either the initial or confirmatory assay in the presence or ab sence of S9 mix.
14. CONCLUSION No substantial increases in the revertant colony counts were obtained with any strain following exposure to the test substance in either the ini tial or confirmatory assa ys in the absence or presence of S9 mix. It is concluded that did not show any evidence of genotoxic activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.Testing Facility Study No.

Page 20 Table 1 - Initial Test in the Absence of S9 Mix Number of revertants Plate observations * Strain Conc. (μg/plate) S9 x 1 x 2 x 3 mean SD x 1 x 2 x 3 Fold response Ü TA1535 Air 0 16 16 19 17 2 - - - 1.0 1.00 0 25 20 17 21 4 - - - 1.2 3.16 0 25 20 22 22 3 - - - - 10.0 0 17 15 18 17 2 - - - 1.0 31.6 0 17 20 28 22 6 - - - 1.3 ~100 0 19 20 16 18 2 - - - 1.1 TA1537 Air 0 12 6 8 9 3 - - - 1.0 0.316 0 8 17 7 11 6 - - - 1.2 1.00 0 12 6 11 10 3 - - - 1.1 3.16 0 11 11 13 12 1 - - - 1.3 10.0 0 10 6 13 10 4 - - - 1.1 31.6 0 7 8 4 6 2 - - - 0.7 ~100 0 7 3 4 5 2 - - - 0.5 T TA98 Air 0 42 27 19 29 12 - - - 1.0 0.316 0 20 21 15 19 3 - - - 0.6 1.00 0 33 22 34 30 7 - - - 1.0 3.16 0 38 28 24 30 7 - - - 1.0 10.0 0 28 27 25 27 2 - - - 0.9 31.6 0 31 22 20 24 6 - - - 0.8 ~100 0 10 6 10 9 2 - - - 0.3 T TA100 Air 0 108 113 117 113 5 - - - 1.0 1.00 0 116 119 128 121 6 - - - 1.1 3.16 0 118 106 135 120 15 - - - 1.1 10.0 0 96 119 120 112 14 - - - 1.0 31.6 0 120 103 118 114 9 - - - 1.0 ~100 0 89 83 106 93 12 - - - 0.8 WP2 uvrA Air 0 57 58 80 65 13 - - - 1.0 1.00 0 62 63 65 63 2 - - - 1.0 3.16 0 63 70 43 59 14 - - - 0.9 10.0 0 52 67 63 61 8 - - - 0.9 31.6 0 58 56 52 55 3 - - - 0.9 ~100 0 53 56 47 52 5 - - - 0.8 * Comments on the plate or background lawn, if applicable: incomplete lawn (IL), no lawn (NL), precipitate (ppt). Ü Fold response in mean revertants co mpared to concurrent vehicle control. SD Sample standard deviation (note that SDs based on two values may be unreliable). T If noted, toxic as indicated by low revertant colony counts (fold response < 0.6), or incomplete/ no background lawn (no meaningful count results for plates with IL or NL). A If noted, low count considered to be due to normal variation rather than toxicity since not clearly dose-related and not outside normal limits based on historical control values.Testing Facility Study No.

Page 21 Table 2 - Initial Test in the Presence of S9 Mix Number of revertants Plate observations * Strain Conc. (μg/plate) S9 x 1 x 2 x 3 mean SD x 1 x 2 x 3 Fold response Ü TA1535 Air + 17 17 26 20 5 - - - 1.0 0.316 + 12 16 11 13 3 - - - 0.7 1.00 + 22 19 26 22 4 - - - 1.1 3.16 + 20 22 26 23 3 - - - 1.1 10.0 + 24 20 17 20 4 - - - 1.0 31.6 + 12 17 13 14 3 - - - 0.7 ~100 + 16 8 7 10 5 - - - 0.5 T TA1537 Air + 10 11 7 9 2 - - - 1.0 1.00 + 10 8 15 11 4 - - - 1.2 3.16 + 6 12 11 10 3 - - - 1.0 10.0 + 18 9 6 11 6 - - - 1.2 31.6 + 7 4 8 6 2 - - - 0.7 ~100 + 9 6 4 6 3 - - - 0.7 TA98 Air + 35 62 40 46 14 - - - 1.0 0.316 + 22 24 22 23 1 - - - 0.5 A 1.00 + 42 47 40 43 4 - - - 0.9 3.16 + 49 28 37 38 11 - - - 0.8 10.0 + 44 39 35 39 5 - - - 0.9 31.6 + 44 27 36 36 9 - - - 0.8 ~100 + 13 20 18 17 4 - - - 0.4 T TA100 Air + 117 139 126 127 11 - - - 1.0 1.00 + 139 169 126 145 22 - - - 1.1 3.16 + 147 134 117 133 15 - - - 1.0 10.0 + 107 126 124 119 10 - - - 0.9 31.6 + 113 122 115 117 5 - - - 0.9 ~100 + 71 85 82 79 7 - - - 0.6 WP2 uvrA Air + 91 96 78 88 9 - - - 1.0 0.316 + 57 44 36 46 11 - - - 0.5 A 1.00 + 67 65 48 60 10 - - - 0.7 3.16 + 80 65 63 69 9 - - - 0.8 10.0 + 68 80 43 64 19 - - - 0.7 31.6 + 70 62 70 67 5 - - - 0.8 ~100 + 47 39 33 40 7 - - - 0.4 T For explanatory footnotes, please see Table 1.Testing Facility Study No.

Page 22 Table 3 Positive Controls for the Initial Test Conc. Number of revertants Fold Strain Treatment (μg/plate) S9 x 1 x 2 x 3 mean SD response Ü TA1535 NaAz 0.5 0 303 371 310 328 37 19 TA1537 9AC 50 0 384 380 308 357 43 41

TA98 2NF 1 0 193 172 171 179 12 6.1 TA100 NaAz 0.5 0 473 520 485 493 24 4.4 WP2 uvrA NQO 0.5 0 227 253 230 237 14 3.6

TA1535 2AA 5 + 522 495 479 499 22 25 TA1537 BaP 5 + 107 113 97 106 8 11

TA98 BaP 5 + 411 422 417 417 6 9.1 TA100 BaP 5 + 1139 984 1003 1042 85 8.2 WP2 uvrA 2AA 15 + 239 227 248 238 11 2.7 Ü Fold response in mean revertants co mpared to concurrent vehicle control. SD Sample standard deviation (note that SDs based on two values may be unreliable).Testing Facility Study No.

Page 23 Table 4 - Confirmatory Test in the Absence of S9 Mix Number of revertants Plate observations * Strain Conc. (μg/plate) S9 x 1 x 2 x 3 mean SD x 1 x 2 x 3 Fold response Ü TA1535 Air 0 20 9 18 16 6 - - - 1.0 6.4 0 22 15 8 15 7 - - - 1.0 12.8 0 15 18 28 20 7 - - - 1.3 25.6 0 15 12 12 13 2 - - - 0.8 51.2 0 18 20 11 16 5 - - - 1.0

~ 100 0 12 15 17 15 3 - - - 0.9 TA1537 Air 0 14 5 9 9 5 - - - 1.0 6.4 0 12 11 11 11 1 - - - 1.2 12.8 0 10 9 10 10 1 - - - 1.0 25.6 0 11 6 5 7 3 - - - 0.8 51.2 0 8 9 7 8 1 - - - 0.9 ~ 100 0 12 9 6 9 3 - - - 1.0 TA98 Air 0 35 22 29 29 7 - - - 1.0 6.4 0 31 25 28 28 3 - - - 1.0 12.8 0 23 26 40 30 9 - - - 1.0 25.6 0 22 24 17 21 4 - - - 0.7 51.2 0 14 20 15 16 3 - - - 0.6 ~ 100 0 18 23 26 22 4 - - - 0.8 TA100 Air 0 121 124 118 121 3 - - - 1.0 6.4 0 105 117 95 106 11 - - - 0.9 12.8 0 120 106 137 121 16 - - - 1.0 25.6 0 110 123 103 112 10 - - - 0.9 51.2 0 92 91 100 94 5 - - - 0.8 ~ 100 0 86 100 92 93 7 - - - 0.8 WP2 uvrA Air 0 49 54 61 55 6 - - - 1.0 6.4 0 39 64 42 48 14 - - - 0.9 12.8 0 71 38 71 60 19 - - - 1.1 25.6 0 53 46 69 56 12 - - - 1.0 51.2 0 43 63 43 50 12 - - - 0.9 ~ 100 0 54 50 51 52 2 - - - 0.9 For explanatory footnotes, please see Table 1.Testing Facility Study No.

Page 24 Table 5 - Confirmatory Test in the Presence of S9 Mix Number of revertants Plate observations * Strain Conc. (μg/plate) S9 x 1 x 2 x 3 mean SD x 1 x 2 x 3 Fold response Ü TA1535 Air + 20 17 12 16 4 - - - 1.0 6.4 + 22 12 17 17 5 - - - 1.0 12.8 + 17 20 16 18 2 - - - 1.1 25.6 + 12 27 17 19 8 - - - 1.1 51.2 + 14 16 17 16 2 - - - 1.0 ~ 100 + 14 16 15 15 1 - - - 0.9 TA1537 Air + 9 3 9 7 3 - - - 1.0 6.4 + 11 8 11 10 2 - - - 1.4 12.8 + 5 6 7 6 1 - - - 0.9 25.6 + 20 9 11 13 6 - - - 1.9 51.2 + 12 8 8 9 2 - - - 1.3 ~ 100 + 11 5 12 9 4 - - - 1.3 TA98 Air + 45 33 38 39 6 - - - 1.0 6.4 + 53 38 32 41 11 - - - 1.1 12.8 + 50 40 37 42 7 - - - 1.1 25.6 + 42 34 42 39 5 - - - 1.0 51.2 + 36 43 40 40 4 - - - 1.0 ~ 100 + 42 49 26 39 12 - - - 1.0 TA100 Air + 103 118 119 113 9 - - - 1.0 6.4 + 108 109 116 111 4 - - - 1.0 12.8 + 139 114 128 127 13 - - - 1.1 25.6 + 127 119 95 114 17 - - - 1.0 51.2 + 91 115 100 102 12 - - - 0.9 ~ 100 + 97 110 105 104 7 - - - 0.9 WP2 uvrA Air + 77 66 45 63 16 - - - 1.0 6.4 + 78 60 52 63 13 - - - 1.0 12.8 + 77 83 72 77 6 - - - 1.2 25.6 + 60 64 57 60 4 - - - 1.0 51.2 + 81 55 50 62 17 - - - 1.0 ~ 100 + 71 54 57 61 9 - - - 1.0 For explanatory footnotes, please see Table 1.Testing Facility Study No.

Page 25 Table 6 Positive Controls for the Confirmatory Test Conc. Number of revertants Fold Strain Treatment (μg/plate) S9 x 1 x 2 x 3 mean SD response Ü TA1535 NaAz 0.5 0 362 360 338 353 13 23 TA1537 9AC 50 0 455 201 490 382 158 41

TA98 2NF 1 0 173 183 141 166 22 5.8 TA100 NaAz 0.5 0 590 599 590 593 5 4.9 WP2 uvrA NQO 0.5 0 243 256 270 256 14 4.7

TA1535 2AA 5 + 503 487 568 519 43 32 TA1537 BaP 5 + 89 76 105 90 15 13

TA98 BaP 5 + 334 352 379 355 23 9.2 TA100 BaP 5 + 1137 1015 1037 1063 65 9.4 WP2 uvrA 2AA 15 + 168 198 182 183 15 2.9 Ü Fold response in mean revertants co mpared to concurrent vehicle control. SD Sample standard deviation (note that SDs based on two values may be unreliable).Testing Facility Study No.

Page 26 Figure 1 TA1535 Revertant Colony Count Historical Results Means of triplicate plate counts from non-GLP and QA-audited GLP studies from 11 March 2005 to 21 April 2009. The grand mean is 22 and SD is 5.



### 2 6 17 41 74 117 94 75 54 38
8 3 3 7 11 1111 112

0 20 40 60 80 100 120 140

<5 8 12 16 20 24 28 32 36 40 44 48 52 Revertant colony countFrequency

Figure 2 TA1537 Revertant Colony Count Historical Results Means of triplicate plate counts from non-GLP and QA-audited GLP studies from 11 March 2005 to 21 April 2009. The grand mean is 15 and SD is 4.



### 1 3 10 54 108 153 127
89 63
21 16 5 4 1 2 1 1 0 20 40 60 80 100 120 140 160 180

<3 6 10 14 18 22 26 30 34 38 Revertant colony countFrequencyTesting Facility Study No.

Page 27 Figure 3 TA98 Revertant Colony Count Historical Results Means of triplicate plate counts from non-GLP and QA-audited GLP studies from 11 March 2005 to 21 April 2009. The grand mean is 35 and SD is 9.

3 3 13 34 110 101 115 95 81 63

23 15 2 2 1 1 0 20 40 60 80 100 120 140

<9 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 >72 Revertant colony countFrequency

Figure 4 TA100 Revertant Colony Count Historical Results Means of triplicate plate counts from non-GLP and QA-audited GLP studies from 11 March 2005 to 21 April 2009. The grand mean is 145 and SD is 20.



### 1 7 14 41 86 135 141

98 73
35 18 8 6 2 0 20 40 60 80 100 120 140 160

<61 80 100 120 140 160 180 200 220 Revertant colony countFrequencyTesting Facility Study No.

Page 28 Figure 5 WP2 uvrA Revertant Colony Count Historical Results Means of triplicate plate counts from non-GLP and QA-audited GLP studies from 11 March 2005 to 21 April 2009. The grand mean is 51 and SD is 12.



### 1 10 14 17 21 62 83 113 101 84 77

42
16 8 1 0 20 40 60 80 100 120

<10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 >90 Revertant colony countFrequencyTesting Facility Study No.

Page 29 Appendix 1 Certific ate of AnalysisTesting Facility Study No.

Page 30Testing Facility Study No.

Page 31 Appendix 2 Historical Positive Control ResultsTesting Facility Study No.

Page 32 Appendix 2 Historical Positive Control Results Mean of the mean results of triplicate plate counts from non-GLP and QA-audited GLP studies from 11 March 2005 to 21 April 2009. Strain Treatment Dose μg/plate S9 Mean SD Range TA1535 NaAz 0.5 0 308 45 150 - 653 TA1537 9AC 10 a 0 1633 1094 234 - 4678 50 b 0 328 165 28 - 1009 TA98 2NF 1 0 154 37 41 - 247 TA100 NaAz 0.5 0 524 71 246 - 1012 WP2 uvrA NQO 0.5 0 658 511 75 - 1495

TA1535 2AA 5 + 342 79 108 - 700 TA1537 BaP 5 + 114 25 34 - 190

TA98 BaP 5 + 378 72 178 - 550 TA100 BaP 5 + 1042 214 404 - 1665 WP2 uvrA 2AA 15 + 330 120 109 - 666
Where a and b indicate standard dose levels for th e pre-incubation and plate incorporation versions of the test, respectivelyTesting Facility Study No.

Page 33 Appendix 3 Protocol and AmendmentsTesting Facility Study No.

Page 34Page 35Page 3 Testing Facility Study No.
1. STUDY SPECIFIC DETAILS Purpose To evaluate the genotoxicity of the test substance using the bacteria l mutation test. Sponsor's Representative

Testing Facility

Compliance Statement This study will be cond ucted 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 testatmosphere 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 471 for the testing of Testing Facility Study No.

Page 36Page 4 Testing Facility Study No. chemicals (1997), the US EPA health effects test guidelines (1998, OPPTS 870.5100) 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 Lot number Expiry date 30 June 2011 Description Colorless gas 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 Assumed to be 100%; all concentra tions to be expressed in term s of material as is Vehicle/diluent Ambient labo ratory air Test Atmosphere Preparation Appropriate mixture prepared in s itu immediately at the start of exposure Stability of Test Atmosphere Not assessed in this study. Formulation Analysis for Achieved Concentratio n Not assessed in this study. Testing Facility Study No.

Page 37Page 5 Testing Facility Study No. Study Design Number of replicates Dose number Material Exposure conc. % v/v 0S9 +S9 Number of strains Initial test a 0 Vehicle - 3 3 5 1 Test substance 0.032 3 3 5 2 0.100 3 3 5 3 0.316 3 3 5 4 1.000 3 3 5 5 3.16 3 3 5 6 10.0 3 3 5 7 31.6 3 3 5 8 ~100 a 3 3 5 Positive control ‡ 3 3 5 CE 20 3 3 5 Confirmatory test b 0 Vehicle - 3 3 5 1 Test substance 3.2 3 3 5 2 6.4 3 3 5 3 12.8 3 3 5 4 25.6 3 3 5 5 51.2 3 3 5 6 ~100 3 3 5 Positive control ‡ 3 3 5 CE 20 3 3 5

a The maximum practical dose (nominally 100%) will be ac hieved by removing as much air as possible from the bag using a vacuum pump, gassing with test substance, purging the atmosphere then re-gassing b The study design for the confirmatory test is dependent on results obtained in the initial test. In the event of toxicity or apparent treatment-related increases in reve rtant counts, the study design will be discussed with the Study Monitor and altered by protocol amendment, if necessary. c Standard positive controls dosed as solutions; concentration dependant on test organism and positive control
Modifications and Notes Gas testing procedures as per A ppendix 3 of Test Facility SOP Sponsor report number: Testing Facility Study No.

Page 38Page 6 Testing Facility Study No. 2. REASON FOR CHOICE OF TEST SYSTEM The bacterial mutation test detects point mutatio ns, which are the cause of many huma n genetic diseases and can play a role in tumor initiation and development.
3. REASON FOR DOSE LEVEL S ELECTION Typically, the test substance is dosed at a range of concentrations but is only assessed at the five highest levels below the toxic le vel or, if non-toxic, at five le vels up to the standard lim it or maximum practical concentration, as appropriate. The highest dose for the initial test is the maximum practical.
4. PARTICIPATING PERSONNEL Study Director

Participatin g Scientist(s)

Assistant Scientist Testing Facility Study No.

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5. SUGGESTED SCHEDULE

Experimental start (first day of exposure of test system to test substance) 12 March 2010
Results (provisional experimental completi on) 29 March 2010 Draft report (non-audited) 16 April 2010 Suggested date for client comments Within 2 weeks of receipt of draft report (see below) Final report May 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.
6. COMPUTER SYS TEMS

• Revertant colony counts are rou tinely collected and saved di rectly into an electronic database using a ProtoCOL automat ed colony counter (version 3.06 supplied by Synbiosis) which minimizes potential bias and transcription errors.

• Results from ProtoCOL are saved and archived as ProtoCOL database files with backed up versions in Microsoft Excel 2000/2003 file form at.

• Results are tabulated, mean values and sam ple standard deviations ca lculated (if required) using Microsoft Excel 2000/2003. Testing Facility Study No.

Page 40Page 8 Testing Facility Study No. 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 The vehicle/diluent will be ambient laborator y air.
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. Unless specifically requested by the Sponsor, all test substanc e remaining at study completion will be returned 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. Gaseous positive control The positive control will be mixed in appropriate proportions with ambient laboratory air in the treatment bags at dosing. Testing Facility Study No.

Page 41Page 9 Testing Facility Study No. Identity: Chloroethane (CE) Vehicle: Ambient laboratory air Concentration: 20% v/v (positive in stra ins TA1535 and WP2 uvrA and weak/borderlin e positive in TA100) 7.5. Standard Positive Controls Sodium azide, 9-aminoacridine, 2-nitrofluoren e, 4-nitroquinoline N-oxide, 2-am inoanthracene and benzo[a]pyrene will be used as positive control substances. Appropriate details will be presented in the report. The positive control formulations will not be subjected to analysis for safety reasons and because the biolog ical response of the test system is considered to be the best measure of the appropriateness of the formulations.
8. EXPERIMENTAL PROCEDURES 8.1. Test system The bacte ria were originally supplied by Molto xTM, NC, USA. Each bacterial culture is identified by its name (TA1535, TA1537, TA98, TA100, WP2 uvrA ) and batch number. Each batch of frozen bacteria is tested for appropriate phenotype characteristics, spontaneous reversion rate and response to diagnostic mutagens. Th e following bacterial strains will be employed: S. typhimurium TA1535 his G46 rfa Δ uvrB S. typhimurium TA1537 his C3076 rfa Δ uvrB S. typhimurium TA98 his D3052 rfa Δ uvrB pKM101 S. typhimurium TA100 his G46 rfa Δ uvrB pKM101 E. coli WP2 trp uvrA The batch identification for each st rain used will be reco rded in the raw data. Fresh bacterial cultures, labeled with initiation da te, study and strain number, will be prepared and will be in the late-log phase of growth at the tim e of use. The density of the cultures will be confirmed to be ≥ 1000 × 10 6 bacteria/mL using a bacterial counting cham ber before the cultures are used in the test. 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 Testing Facility Study No.

Page 42Page 10 Testing Facility Study No. Sprague-Dawley rat liver fraction supplied by Molt ox) and the following ster ile cofactors: 8 mM MgCl 2 , 33 mM KCl, 100 mM sodium phosphate buffe r pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP. The S9 mix will be stored at ca. 4°C or on ice until required. A copy of the manufacturer’s quality control certificate(s) for the S9 fraction will be retained as raw data.
8.3. Sterility and vehicle controls The ster ility of the buffer, S9 mix, and positive co ntrols (high dose where more than one dose is tested) will be assessed on the day of the test using appropriate prepara tions without bacteria. The spontaneous m utation rates of the bacterial stra ins will be assessed us ing concurrent control samples in which the bacteria are exposed to the vehicle. W here an unusual vehicle is used, a set of untreated (negative) control pl ates may be included to assess pot ential effects of the vehicle – refer to the study design.
8.4. Plate Incorporation Method A 0.5 mL aliquot of S9 mix (+S9) or phosphate buffer 0.2M pH 7.4 (0S9) will be combined with 0.1 mL bacterial culture in a ster ile container. Where appropriate , a standard volum e (0.1 mL) of the test positive control will be added. Approxi mately 2 mL of molten top agar supplemented with 0.05 mM biotin and minimal (0.05 mM) his tidine and minimal (0.05 mM) tryptophan will be added immediately afterward. The solution will be mixed and overlaid onto a minimal glucose plate (1.5% agar, Vogel- Bonner medium E, 2% glucose). After the overlay solidifies, the plates will be inverted and incubated at ca. 37°C for 48 to 72 h. The sterile containers and each plate will be labeled with: study number, a unique number that identifies the contents by reference to the experimental de sign held with the raw data, and the day code. After the overlay solidifies, the plates will be inverted, stacked and placed into Tedlar bags (or equivalent) that have been slit on one side (one bag per treatment). The bags will be sealed with adhesive tape and the air inside the bags (hi gh dose only) will be evacuated. Inside a fumehood or laminar hood, appropriate volumes of ambient laboratory air, stock test gas or positive control g as will be added via the se ptum/valve into the bag to achieve the final concentrations indicated in the Study design. For th e highest dose of the test gas, the gas will be added to the bag, then purged prior to the addition of a second volume of gas. The plates in the bags will be transferred to incubators atca. 37°C for 48 to 72 h. After the treatment period, th e bags will be opened in a flame hood or laminar hood and the plates equilibrated in the hood for ca. 20 mins before counting colonies. Testing Facility Study No.

Page 43Page 11 Testing Facility Study No. The sterility plates for the buffer and S9 mix as well as the plates treated with the routine liquid positive controls will not be placed into Tedlar bags . They will be inverted and incubated at ca. 37°C for the same duration as the test atmosphere-treated plates.
9. EVALUATION AND I NTERPRETATION OF RESULTS After the incubation period, the plat es will be examined visually a nd, if necessary, with the aid of an inverted mi croscope. Plates will be evaluated for the qualityof the background lawn and the number of revertant colonies. Revertant colony counts are routinel y collected and saved directly into an electronic database using a ProtoCOL automated colony counter (version 3.06 supplied by Synbiosis). The electronic raw data will be tr ansferred to CD-ROM and held with the rest of the study data. Colony numbers will be enumeratedvisually if precipita tion or other artifacts interfere with the colony counter (if practical), or at the Study Di rector’s discretion. Any visual counts and the presence of visi ble precipitate are manually recorded in the raw data. If precipitation inte rferes with observation of the background lawn, this is recorded and noted in the report. In such cases, the background lawn is assumed to be norm al if the colony counts are similar to the vehicle control. In the rare even t that precipitation preven ts counting of revertant colonies, this is noted in the report and the colony count is considered not readable. In the event of an apparent tr eatment-related increase in colony num bers for the test substance, the Study Director may authorize replica plati ng to confirm the phenot ype of colonies to preclude potential artifacts. If available, the plates from at least five non-toxic dose levels of the test substance will be assessed in each experiment, i.e. the five highe st levels below the toxic level. If the test substance is nontoxic, then the five levels up to the highest will be assessed and reported. Toxic effects of the test substance are normally indi cated by the partial or complete absence of a background lawn (colony counts, if any, will not be reported) or a substantial dose-related reduction in revertant colony count s compared with lower dose le vels and concurrent vehicle controls (i.e. fold response < 0.6) taking into account the laboratory hi storical control range. Means and standard deviations for appropriate do se levels will be calculated using Microsoft ® Excel 2003 or 2007 and presented in the report together with laboratory historical control information. The mean number of revertant col onies for all treatment groups will be compared with those obtained for the concurrent vehicle control group. The mutageni c activity of the test substance is routinely assessed by applying the following criteria: Testing Facility Study No.

Page 44Page 12 Testing Facility Study No. 1. The results will be considered positive (i.e. i ndicative of mutagenic potential) if both the following criteria are met.

● The results for the test substance show a substantial increase in revertant colony counts, i.e. response ≥ 2 times the concurrent vehi cle control values for T A98, TA1535, TA1537 and WP2 uvrA , or ≥ 1.5 times for TA100, with mean value(s) outside the laboratory histor ical control range (beyond the 98% tolerance limit). Otherwise results are considered negative.

● The above increase mustbe dose related an d/or reproducible i.e. increases must be obtained at more than one experiment al point (more than one strain or dose level, more than one occasion or with different methodologies). 2. If the second criterion is not met, fur ther te sting may be appropriate to clarify equivocal results using an appropriately modified study design, e.g. a narrower dose interval with the appropriate strain. In such cases, if no substantial increase is obtained in the confirmatory test, the results will be considered negative. For an assay to be considered valid, the mean revertant colony counts of the vehicle/negative controls for each strain should lie close to or wi th in the current histori cal control range of the laboratory. All positive control s ubstances (with S9 where required) should produce increases in revertant colony numbers to at least twice the concurrent vehi cle control levels with the appropriate bacterial strain (1.5× for strain TA100). Invalid assa y results will normally lead to an automatic repeat of the appropriate part of the assay. If that is not pr actical then any values that do not comply with these criteria will be discussed in the report.
10. QUALITY ASSURANCE The Quality Assurance department of will undertake and document inspections and audits of critical phases of the study performed 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.
11. 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: Testing Facility Study No.

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• All raw data

• Documents generated at during this study

• The original copy of the protocol (including amendments, if any)

• The final report Subsequent disposition will be docu m ented at the Testing Facility.
12. 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
13. 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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