# Final Report

Study Title Determination of the IR, UV/Vis, Mass and NMR spectra, Purity Assessment and Identification of Impurities

Study Director Ben Foster

Authors Ben Foster Matthew Crane

Sponsor

Study Monitor Warren Scott CSI – Europe Pentlands Science Park, Penicuik, Near Edinburgh, EH26 0PZ

Test Facility Smithers Viscient (ESG) Ltd. Otley Road, Harrogate North Yorkshire, HG3 1PY UNITED KINGDOM

Study Number 8260178

Report Issued December 2012

Page Number 1 of 90Study Number 8260178 Final Report

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### RESPONSIBLE PERSONNEL

Determination of the IR, UV/Vis, Mass and NMR spectra, Purity Assessment and Identification of Impurities

The following personnel were responsible for key elem ents of the study:

STUDY MANAGEMENT NAME Study Director B Foster Scientific Reviewer C Cooke Study Co-ordinator M Crane

OPERATIONAL SUPERVISION Analysts D Thomas C Wilson B Morrison (mass spectrometry) Dr S J Byard (NMR)

QUALITY ASSURANCE Senior Manager, Quality Assurance (Covance Laboratories Ltd. 20 Jan 2012 to 31 May 2012) L Gillbanks

Senior Director, Regulatory Affairs and Quality Systems (Smithers Viscient 01 June 2012 to present) K SmithStudy Number 8260178 Final Report

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### ARCHIVE STATEMENT

Determination of the IR, UV/Vis, Mass and NMR spectra, Purity Assessment and Identification of Impurities

All primary data, or auth enticated copies thereof, specimens or samples will be retained in the Covance Laboratories Limite d archives, on behalf of Smithers Viscient (ESG) Limited, for ten years af ter issue of the final report. At this time, the Sponsor will be contacted to determine whether th e data should be returned, retained or destroyed on their behalf. Sponsors will be notified of the financ ial implications of each of these options at th at time. One copy of the protocol and final report respectively will be held according to procedures described in local SOPs.

STUDY TIMETABLE
This study was initiated on 20 January 2012 by the signing of the study protocol. Experimental work started on 20 January 2012 with the commencem ent of NMR analysis and finished on 12 April 2012 with the assignment of absorption peaks for the determination of IR spectra. Study comple tion is defined as the date of signing of the final report by the Study Director. On 1 June 2012, the test facility was acquired by Smithers Viscient LLC., becoming the legal entity Smithers Viscient (ESG) Ltd.Study Number 8260178 Final Report

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### CONTENTS

STUDY DIRECTOR AUTHENTICATION AND GLP COMPLIANCE STATEMENT ..................... 2

QUALITY ASSURANCE STATEMENT............................................................................................... 3

REVIEWING SCIENTIST'S STATEMENT........................................................................................... 4

RESPONSIBLE PERSONNEL ..........................................................................................................

..... 5

ARCHIVE STATEMENT ..............................................................................................................

......... 6

STUDY TIMETABLE................................................................................................................

............. 6

CONTENTS............................................................................................................................................. 7

SUMMARY ........................................................................................................................

..................... 9

INTRODUCTION.................................................................................................................................. 11

OBJECTIVE ......................................................................................................................

.................... 11

PROCEDURES.....................................................................................................................

................. 11 Protocol Adherence......................................................................................................................... 11 Infrared Absorptio n Spectroscopy...............................................................................................

.... 11 Ultraviolet-Visibl e Spectroscopy ...............................................................................................

..... 12 Mass Spectro metry.......................................................................................................................... 12 Nuclear Magnetic Res onance Spectroscopy ................................................................................... 13 Analytical Procedure for

.............................. 13 Analytical Procedure for identification and analysis of impurities and determination of purity .................................................................... 14 Analysis of Test Substance Purity..............................................................................................

..... 14 Analysis of Test Subs tance for Im purities......................................................................................

. 14

RESULTS ........................................................................................................................

...................... 15 Infrared Absorptio n Spectroscopy...............................................................................................

.... 15 Ultraviolet-Visibl e Spectroscopy ...............................................................................................

..... 15 Mass Spectro metry.......................................................................................................................... 16 Nuclear Magnetic Res onance Spectroscopy ................................................................................... 16 Analytical Procedure 16 Analytical Procedure for Identifica tion and Analysis of Impurities................................................ 17 Analysis of Test Substance Purity..............................................................................................

..... 17 Analysis of Test Subs tance for Im purities......................................................................................

. 17

CONCLUSION ...................................................................................................................................... 18

TABLES.........................................................................................................................

........................ 20 Table 1 Infrared spectral details .............................................................................................

........ 21 Table 2 Ultraviolet-visible spectral character istics,

.................................................................................................................................... 22 Table 3 Preci sion results .....................................................................................................

........... 23 Table 4 Accuracy (recovery) results ...........................................................................................

... 24 Table 5 Determination of test subs tance purity and impurity content............................................ 25Study Number 8260178 Final Report

- 8 - FIGURES ............................................................................................................................................... 26 Figure 1 Structure of

.................................. 27 Figure 2 Infrared spectru m between 4000 and 2000 cm

.............................................................. 28 Figure 3 Infrared spectrum between 2000 and 400 cm -1

................................................................ 29 Figure 4 UV / Visible spectrum for neutral solution in methanol .................................................. 30 Figure 5 UV / Visible spectrum for acidic solution in methanol.................................................... 31 Figure 6 UV / Visible spectrum for basic solution in methanol..................................................... 32 Figure 7 UV / Visible spectrum for basic solution in methanol after neutralisation ...................... 33 Figure 8 Positive ion mass sp ectra for

........................................................... 34 Figure 9 Proposed fragmentation pathway for

............................................... 35 Figure 10 Negative ion mass spectra for

............................................................ 36 Figure 11 Proposed fragmenta tion pathway for

................................................. 37 Figure 12 Analytical procedur e – chromatogram for acetonitrile .................................................. 38 Figure 13 Analytical procedure – chroma togram for 1.0 μg/mL calibration standard................... 38 Figure 14 Analytical procedure – chroma togram for 10 μg/mL calibration standard.................... 39 Figure 15 Analytical procedure – chroma togram for 100 μg/mL calibration standard.................. 39 Figure 16 Analytical procedure – chro matogram for 2 μg/mL recovery sample ........................... 40 Figure 17 Analytical procedure – chro matogram for 50 μg/mL recovery sample ......................... 40 Figure 18 Analytical procedure – chro matogram for 95 μg/mL recovery sample ......................... 41 Figure 19 Analytical pr ocedure - calib ration line........................................................................... 42 Figure 20 Purity and content of impuritie s analysis – chromat ogram for acetonitrile ................... 43 Figure 21 Purity and content of impurities analysis – chromatogram for

................ 43 Figure 22 Purity and content of impurities analysis – chromatogram for

................ 44 Figure 23 Purity and content of impuritie s analysis – chromat ogram for ..................... 44 Figure 24 Purity and content of impurities an alysis – chromatogram for test substance ............... 45

APPENDICES........................................................................................................................................ 46 Appendix 1 Certificate of Analysis............................................................................................

.... 47 Appendix 2 NMR Phase Report...................................................................................................

.. 48 Appendix 3 Analytical Proc edure CLE (PC) 82601 78-01D .......................................................... 79 Appendix 4 Analytical Proc edure CLE (PC) 82601 78-02D .......................................................... 85Study Number 8260178 Final Report

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### SUMMARY
The following physico-chemical properties of were evaluated by the in dicated techniques:

Property Test method Results
IR Spectra n/a Obtained spectrum was consistent with expected structure.
UV Spectra OECD 101 Conditions Maximum absorbance wavelength (nm) Extinction coefficient ( ε ) Acidic

The spectra and associated data are very similar under the neutral and acidic conditions, indicating no change in conformation. In basic solution, three absorbance maximawere observed at The difference in the UV spectrum under basic conditions is considered to result from an irreversible change such as hydrolysis or degradation rather than a reversible conformational change, e.g. an ionisation. Upon ne utralisation of the basic solution, the absorbance spectrum was seen to have changed from that recorded previously, supporting the hypothesis of an irreversible change. This finding is consistent with the structure of the which indicates no groups with appreciable ac idic or basic character.

Mass Spectra n/a Obtained spectra were consistent with expected structure.
NMR Spectra n/a Obtained spectra were consistent with expected structure. Analytical procedure: Accuracy and Precision A high performance liquid chromatography with ultra violet detection (HPLC-UV) method for determination of was successfully validated. Precision and accuracy were assessed by injections of test substance solutions prepared in acetonitrile. Conc (μg/mL) Mean Recovery (%) Precision (%RSD) n

2 94 0.80 3 50 100 0.18 3 95 99 0.22 3

Linearity Response of the HPLC-UV syst em to

was linear, over the range 1 to 100 μ g/mL, with a correlation coefficient (r) of 0.999976 and a calibration curve formula of Y=1830 + 10700X. The linearity of detector response was therefore established. Specificity SANCO/3030/99 rev 4

All solutions were free of any co mponents that interfered with the analysis of to The method was therefore considered specific for toStudy Number 8260178 Final Report

- 10 - Property Test method Results Analytical procedure for analysis of purity and impurities SANCO/3030/99 rev 4 No method validation was carried out for this test as no reference material was available for the test substance. Results were calculated based on percent peak area. Purity analysis n/a 99.5% Analysis for impurities n/a = 0.5%Study Number 8260178 Final Report

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### INTRODUCTION

The test substance, was allocated the code ESTS 06/12 fo r reference throughout the study.

Name:

Chemical Structure:

CAS Number: Chem ical Formula: Molecular Weight: Physical State: White crystalline powder Sponsor Batch: 12166 Purity: 99.5% 1 

### Arrival Date: 11 Jan 2012 Expiry Date: 22 March 2013 1 

### Storage Conditions: Room temperature (15-30°C)

A copy of the Certificate of Analysis generated during this study is shown in Appendix 1

.

OBJECTIVE
The objective of this study is to evaluate the IR, UV/Vis, Mass and NMR spectra, of the test substance and to develop and validate analytical me thods for purity assessment and for identification of impur ities for the test substance, .

PROCEDURES

Protocol Adherence The study was performed in acco rdance with the protocol and three amendments.

Infrared Absorption Spectroscopy Method The test substance was combined with pot assium bromide, ground to a very fine powder, and formed into an IR-trans parent disc under hydraulic pressure.

1 Determined within this studyStudy Number 8260178 Final Report

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### The spectrum was recorded on a Perkin Elmer Spectrum One instrument against a blank potassium bromide disc at a temper ature of 23°C, compiled from 16 scans between 4000 and 400 cm -1 

### at a resolution of 2 cm -1



### . Ultraviolet-Visible Spectroscopy Introduction The experime ntal procedures followed those described in OECD Guideline 101.

Method Methylene Blue solution was used to verify the correct operation of the spectrom eter, with a spectrum recorded between 190 and 750 nm in 10 mm pathlength in a far-UV quartz cell. The absorbance wavelengths obt ained were compared to those in the literature and the spectrometer was consid ered to be operatin g correctly if the recorded wavelengths were within ± 1 nm of the literature values.
The test substance exhibited poor solubility in water, but was adequately soluble in methanol. Methanol was therefore selected as the solvent. Stocks of the test substance were prepared at concentrations of 9 88, 1016 and 1037 μg/mL in methanol (neutral), in methanol containing 10% 1 mol/L hydroc hloric acid (acidic) and in methanol containing 10% 0.999 mol/L sodium hydroxi de solution (basic ), respectively. These stocks were then further diluted to give final sample concentrations of 19.76 and 9.88 μg/mL in methanol (neutral), 20.32 and 10.16 μg/mL in methanol containing 10% 1 mol/L hydrochloric acid (acidic) , 20.74 μg/mL in methanol containing 10% 0.999 mol/L sodium hydroxide solution (basic) and 20.74 μg/mL in methanol containing 9% 0.999 mol/L sodium hydroxide solution and 1% 1 mol/L hydrochloric acid (neutralised, verified by indicator stri ps). Spectra of each preparation were recorded between 190 and 750 nm in 10 mm pathlength far-UV quartz cells against appropriate solvent blanks at 21°C. The in strument used was a Perkin Elmer Lambda 35 spectrophotometer with a slit width of 1 nm, and a scan speed of 120 nm/min.

Mass Spectrometry Method A solution of was supplied at ca 1 mg mL -1 

### this was diluted to ca 1 μg mL in methanol for analysis.

This solutio n was introduced into the mass spectrometer by infusion at a flow of

ca 10 μL min -1 

### into a flow of methanol of ca 100 μL min -1



### . LC/MS conditions for Instrumentation: Therm oFisher LTQ Orbitrap hybrid mass spectrometer Ionisation Mode: Positive ion electrospray Scan Range: MS: 95-600 m/z, MS 2 

### : data dependant Source Voltage: 4.5 kV Capillary Temperature: 280°C Resolution: MS: 60000, MS 2 

### 7500Study Number 8260178 Final Report

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### LC/MS conditions for Instrumentation: Therm oFisher LTQ Orbitrap hybrid mass spectrometer Ionisation Mode: Negative ion electrospray Scan Range: MS: 115-400 m/z, MS 2 

### : data dependant Source Voltage: 3.5 kV Capillary Temperature: 280°C Resolution: MS: 60000, MS 2 

### 7500

Nuclear Magnetic Resonance Spectroscopy Method Full details of the procedure employed are pr ese nted in the phase report, which can be found in Appendix 2

. Analytical Procedure for The procedure CLE (PC) 8260178-01D for determining the content of was developed in this study. The method used high performance liquid chro matography with ultra-violet detection (HPLC-UV), following dilution with acetonitr ile to obtain an analytical solution. The full procedure can be found in Appendix 3

.
The me thod was assessed against the criter ia detailed in the following sections:
Specificity Test solutions were prepared at appropria te concentration levels along with a blank acetonitrile sam ple to assess freedom from any interfering (co-chromatographic) peaks.

Linearity The analytical calibration covered a range of 1 to 100 μg/mL. The linearity of detector response for standard solutions over th e concentration range (nine different concentrations) was determ ined by least squares linear regression.
Precision (Repeatability) For procedur e CLE (PC) 8260178-01D, the repeatability of the analysis was determined by analysis of six replicates of a prepared sample solution in acetonitrile.
Solutions were prepared at concentrati ons of 2 μg/mL, 50 μg/mL and 95 μg/mL.
The prepared solutions were diluted, as deta iled in the analytical procedure, to fall within the calibration range. The mean RSD (relative standard deviation) was determined.

Accuracy For procedure CLE (PC) 8260178-01D, the recovery of the analysis was determined by analys is of prepared sample soluti ons at each of three concentrations.
Solutions were prepared at concentrati ons of 2 μg/mL, 50 μg/mL and 95 μg/mL.Study Number 8260178 Final Report

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### Each of the prepared solutions was dilute d for HPLC-UV analysis, as detailed in the analytical procedure, to fall within th e calibration range. The mean recovery, and RSD (relative standard deviation) were de termined for each set of preparations.

Analytical Procedure for identificati on and analysis of impurities and determination of purity The procedure CLE (PC) 8260178-02D for determining i mpurities in was developed in this study. The method used high performance liquid chro matography with ultra-violet detection (HPLC-UV), following dilution with acetonitr ile to obtain an analytical solution. The full procedure can be found in Appendix 4

.
No validation was carried out on this me thod due to the fact that no reference material was available for the test substance and th e purity was calculated using percent peak area values. This is a deviation from the pr otocol but due to the nature of the test substance, there is not deemed to be any impact on the validity of the results or the integrity of the study.

Analysis of Test Substance Purity The purity of the test substance was obtained by analysis of test substance solutions using the conditions stated in proc edure CLE (PC) 8260178-02D. Purity was calculated using percent peak area.

Analysis of Test Substance for Impurities Determination of impurities was carried out by the analysis of solutions of test substance,
, and using the conditions stated in procedure CLE (PC) 8260178-02D. Content of impurities was calculated using percent peak area.Study Number 8260178 Final Report

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### RESULTS
Infrared Absorption Spectroscopy Table 1 lists the ma jor peaks and their tenta tive assignments; some minor absorbance bands were not readily assignable.
Assignments were made by consulting the following literature reference:
Spectroscopic Methods in Organic Chemistry - Williams and Fleming 6 th 

### (2008) edition, McGraw-Hill.

The peaks are characteristic of the test subs tance, and are regarded as consistent with the accepted structure, which is shown in Figure 1

. Figure 2 shows the spectrum between 4000 and 2000 cm -1 

### and Figure 3 shows the spectrum between 2000 and 400 cm -1



### . Ultraviolet-Visible Spectroscopy The tested solutions were prepared at a concentration of 19.76 and 9.88 μg/mL in

m ethanol (neutral), 20.32 and 10.16 μg/mL in methanol containing 10% 1 mol/L hydrochloric acid (acidic), 20.74 μg/mL in methanol containing 10% 0.999 mol/L sodium hydroxide solution (basic) and in methanol containing 9% 0.999 mol/L sodium hydroxide solution with 1% 1 mol/L hydrochloric acid (neutralised, verified by indicator strips). The molecular formula of is C 26 

### H 36 

### BNO 6 

### , and the molecular weight used to calculate the molar extinction coefficients (absorbance of a 1 cm path le ngth of a solution containing 1 mole/L of substance) was . The tested mo lar concentrations were therefore:

Neutral conditions - for the 19.76 and 9.88 μg/mL solution s, respectively.

Acidic conditions - for the 20.32 and 10.16 μg/mL solution s, respectively.

Basic conditions - for the 20.74 μg/mL solution.

The test resu lts are shown in Table 2 , and spectra are recorded in Figure 4 to Figure 7

. No significant absorbances, other than some slight noise, were recorded in any of the solvent blank spectra.
The spectra and associated data are very similar under the un adjusted and acidic conditions, indicating no change in conformation. In the acidic solution, were observed. In the neutral solution, were observed. In the basic solution, . The difference in the UV spectrum unde r basic conditions is considered to result from an irreversible change such as hydrolysis or degradation rather than aStudy Number 8260178 Final Report

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### reversible conformational ch ange, e.g. an ionisation. Upon neutralisation of the basic solution, the absorbance spectrum was seen to have changed from that recorded previously, supporting the hypothesis of an irreversible change.

Mass Spectrometry Full scan and MS / 

### MS spectra were produced by infusion of the test substance into the mass spectrometer. The test substance was ionic and in solution formed a . Therefore, two characteristic spectra were generated using positive ion electrospray for the analysis of the nd negative ion elec trospray for analysis of the

Tri-n-butylammonia Full spectru m MS demonstrated a base peak positive ion of m/z which was consistent with the structure of

MS 2 

### fragmentation of m/z yielded a product ion spect rum containing ions of m/z
The mass spectra are shown in Figure 8

. A proposed fragme ntation pathway is shown in Figure 9

.

Full spectru m MS demonstrated a base peak negative ion of m/z , which was consistent with the structure of

MS 2 

### fragmentation of m/z yielded a product ion sp ectrum containing an ion of m/z
The mass spectra are shown in Figure 10

. A proposed fragme ntation pathway is shown in Figure 11

. Nuclear Magnetic Resonance Spectroscopy Full details of the results are presented in the phase report, which can be found in Appendix 2

. Analytical Procedure The me thods were assessed against the crit eria detailed in the following sections:
Specificity All solutions were free of any comp onents that interfered with the analysis of the test substance. The method was therefore cons idered specific for . Example chro matograms of acetonitrile and test substance solutions are presented in Figure 12 to Figure 18

.Study Number 8260178 Final Report

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### Linearity Response of the HPLC-UV system t o was linear, over the range 1 to 100 μ g/mL, with correlation coefficients (r) of 0.999976. The linearity of detector response was therefore established.
A representative calibration plot is presented in Figure 19

. Precision (Repeatability) Repeatability of the me thod, expressed as the relative standard deviation (RSD) measurements for obtained from solutions in acetonitrile, were within the acceptan ce criteria specified in SANCO/3030/99 (rev 4) of <9.34% for the 2 μg/mL 2 

### solution, <5.73% for the 50 μg/mL solution and <5.21% for the 95 μg/mL solution, being 0.80%, 0.18 and 0.22%, respectively. The precisi on data are shown in Table 3 . Example chrom atograms are presented in Figure 16 to Figure 18

. Accuracy (Recovery) Mean recove ries of were within the acceptable ranges of 80 - 120% for the 2 μg/mL recovery level and 90 - 110% for the 50 μg/mL and 95 μg/m L recovery levels. The accuracy data are shown in Table 4
, respectively. Exam ple chromatograms are presented in Figure 16 to Figure 18

. Analytical Procedure for Identifica tion and Analysis of Impurities No peaks were seen for the or standard solutions, which was probably due to lack of UV absorbance for these compounds. A peak was seen for the standard. There were no inte rfering peaks from th e acetonitrile blank.

Analysis of Test Substance Purity Analysis of the test substance using the conditions stated in procedure CLE (PC) 8260178-02D produced two chrom atographic peaks, one of these was the and the other was

. Based on percentage of total peak area, the purity of the test substance was calculated to be 99.5%. The purity data are shown in Table 5

. Analysis of Test Substance for Impurities Analysis of the test substance, , and using the conditions stated in procedure CLE (PC) 8260178-02D gave responses for the test substance and only. The absence of and is probably due to lack of UV absorbance for these compounds. Based on percentage total peak area, the content of in the test substance was calculated to be 0.5%. The impurity data are shown in Table 5 . Chroma tograms for the purity analysis and the analysis for impurities are shown in Figure 20 to Figure 24

.

2 Precision acceptance values were calculated using the modified Horwitz equation as per SANCO guideline

3030/99 (rev 4). See Table 3 for details of ca lculation.
3 Recovery acceptance ranges were taken from SANCO gui deline 3030/99 (rev 4) for the nominal spiked accuracy

concentrations used. See Table 4 for details.Study Number 8260178 Final Report

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### CONCLUSION
The following physico-chemical properties of were evaluated by the indicated techniques:

Property Test method Results
IR Spectra n/a Obtained spectrum was consistent with expected structure.
UV Spectra OECD 101 Conditions Maximum absorbance wavelength (nm) Extinction coefficient ( ε )

The spectra and associated data are very similar under the neutral and acidic conditions, indicatin g no change in conformation. In basic solution three absorban ce maxima were observed at The difference in the UV spectrum under basic conditions is considered to result from an irreversible change such as hydrol ysis or degradation rather than a reversible conformational chan ge, e.g. an ionisation. Upon neutralisation of the basic solution, the absorbance spectrum was seen to have changed from that recorded previously, supporting the hypothesis of an irrevers ible change. This finding is consistent with the structur e of the wh ich indicates no groups with appreciable acidic or basic character. Mass Spectra n/a Obtained spectra were consistent with expected structure.
NMR Spectra n/a Obtained spectra were consistent with expected structure. Analytical procedure: Accuracy and Precision An high performance liquid chromatography with ultra violet detection (HPLC-UV) method for determination of was successfully validated. Precision and accuracy were assessed by injections of test substance solutions prepared in acetonitrile. Conc (μg/mL) Mean Recovery (%) Precision (%RSD) n
2 94 0.80 3 50 100 0.18 3 95 99 0.22 3

Linearity Response of the HPLC-UV syst em to

was linear, over the range 1 to 100 μ g/mL, with a correlation coefficient (r) of 0.999976 and a calibration curve formula of Y=1830 + 10700X. The linearity of detector response was therefore established. Specificity SANCO/3030/99 rev 4

All solutions were free of any components that interfered with the analysis of to . T e method was therefore considered specific for
to ).Study Number 8260178 Final Report

- 19 - Property Results Analytical procedure for analysis of purity and impurities SANCO/3030/99 rev 4 No method validation was carried out for this test as no reference material was available for the test substance. Results were calculated based on percent peak area. Purity analysis n/a 99.5% Analysis for impurities n/a = 0.5%Study Number 8260178 Final Report

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### TABLESStudy Number 8260178 Final Report

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### Table 1 Infrared spectra l detailsStudy Number 8260178 Final Report

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### Table 2 Ultraviol et-visible spectral characteristics,

Conditions Maximum absorbance wavele ngth (nm) Extinction coefficient ( ε )Study Number 8260178 Final Report

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### Table 3 Precis ion results

Sequence ID Sample Concentration found (μg/mL) Mean Precision %RSD

P and A 350 nm QC 2 1.866557
QC 2 1.870157
QC 2 1.872252
QC 2 1.880559
QC 2 1.882450
QC 2 1.908147 1.880020 0.80

QC 50 49.944849
QC 50 50.017947
QC 50 50.052337
QC 50 50.209385
QC 50 50.100282
QC 50 50.088059 50.068810 0.18

QC 95 94.426485
QC 95 94.196354
QC 95 94.316028
QC 95 94.272734
QC 95 94.617583
QC 95 94.712673 94.423643 0.22

Acceptance criteria for precision from SANCO/3030/99 (revisi on 4) are based on the modified Horwitz equation:

%RSD R = 2 (1-0.5 x log C)

Where:

RSD R = among laboratory relative standard deviation C = percent concentration of active ingredient expressed as a decimal (e.g. 100% = 1).

For estimation of repeatability this is modified to:

%RSD r = %RSD R x 0.67

For acceptance of results the precision va lues must be below the corresponding RSD r value.

For QC 2 RSD r = 2 (1-0.5 x log 0.0000025) x 0.67 = 9.34 For QC 50 precision RSD r = 2 (1-0.5 x log 0.000064) x 0.67 = 5.73 For QC 95 precision RSD r = 2 (1-0.5 x log 0.00012) x 0.67 = 5.21Study Number 8260178 Final Report

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### Table 4 Accuracy (recovery ) results

Sequence ID Sample Prepared conc. (μg/mL) Measured

conc. (μg/mL) Recovery (%) Mean recovery

(%) Std dev Precision %RSD

P and A 350

nm QC 2 2 1.866557 93.3278

QC 2 2 1.870157 93.5079
QC 2 2 1.872252 93.6126
QC 2 2 1.880559 94.0208
QC 2 2 1.882450 94.1225
QC 2 2 1.908147 95.4074 94 0.75 0.80

QC 50 50 49.944849 99.8897
QC 50 50 50.017947 100.0359
QC 50 50 50.052337 100.1047
QC 50 50 50.209385 100.4188
QC 50 50 50.100282 100.2006
QC 50 50 50.088059 100.1761 100 0.18 0.18

QC 95 95 94.426485 99.3963
QC 95 95 94.196354 99.1541
QC 95 95 94.316028 99.2800
QC 95 95 94.272734 99.2345
QC 95 95 94.617583 100.5975
QC 95 95 94.712673 100.6976 99 0.21 0.22

Acceptance criteria for precision from SANCO/3030/99 (revision 4) are based on the following confidence intervals for % mean recovery:

80 – 120% for a nominal active i ngredient content of <0.01% 90 – 110% for a nominal active ingred ient content of 0.01% to 0.1%Study Number 8260178 Final Report

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### Table 5 Determination of test substa nce purity and impurity content

Sample Peak area of Peak area of Content of

% by peak area) Content of (% by peak area)

ESTS 06/12 187181404 932547 99.5 0.5Study Number 8260178 Final Report

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### FIGURESStudy Number 8260178 Final Report

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### Figure 1 Structure ofStudy Number 8260178 Final Report

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### Figure 4 UV / Visible spectrum for neut ral solution in methanolStudy Number 8260178 Final Report

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### Figure 5 UV / Visible spectrum for acid ic solution in methanolStudy Number 8260178 Final Report

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### Figure 6 UV / Visible spectrum for ba sic solution in methanolStudy Number 8260178 Final Report

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### Figure 7 UV / Visible spectrum for basic soluti on in methanol after neutralisationStudy Number 8260178 Final Report

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### Figure 8Study Number 8260178 Final Report

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### Figure 10Study Number 8260178 Final Report

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### Figure 12 Analytical procedure – chro matogram for acetonitrile

SampleName: MeCN Date Acquired: 19-Mar-2012 09:22:09 GMT +00:00 Result Id: 2837AU

0.000 0.001 0.002 0.003 0.004

Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0 0



### Figure 13 Analytical procedure – chromatogram for 1.0 μg/mL calibration standard

SampleName: Cal 1 Date Acquired: 19-Mar-2012 10:04:19 GMT +00:00 Result Id: 2829- 7.171AU

-0.001 0.000 0.001 0.002 0.003 0.004

Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0 0Study Number 8260178 Final Report

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### Figure 14 Analytical procedure – chromatogram for 10 μg/mL calibration standard

SampleName: Cal 10 Date Acquired: 19-Mar-2012 18:50:24 GMT +00:00 Result Id: 2836- 7.166AU

0.000 0.010 0.020 0.030 0.040 0.050

Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0 0



### Figure 15 Analytical procedure – chromatogram for 100 μg/mL calibration standard

SampleName: Cal 100 Date Acquired: 19-Mar-2012 09:43:18 GMT +00:00 Result Id: 2828- 7.157AU

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0 0Study Number 8260178 Final Report

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### Figure 16 Analytical procedure – chromatogram for 2 μg/mL reco very sample

SampleName: QC 2 Date Acquired: 19-Mar-2012 16:23:09 GMT +00:00 Result Id: 2852- 7.175AU

-0.001 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009

Minutes 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0 0



### Figure 17 Analytical procedure – chromatogr am for 50 μg/mL recovery sample

SampleName: QC 50 Date Acquired: 19-Mar-2012 13:13:53 GMT +00:00 Result Id: 2845- 7.159AU

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0 0Study Number 8260178 Final Report

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### Figure 18 Analytical procedure – chromatogr am for 95 μg/mL recovery sample

SampleName: QC 95 Date Acquired: 19-Mar-2012 10:25:26 GMT +00:00 Result Id: 2839- 7.154AU

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.0 0Study Number 8260178 Final Report

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### Figure 19 Analytical procedure - calibration lineA rea

-2.0x10 5 0.0 2.0x10 5 4.0x10 5 6.0x10 5 8.0x10 5 1.0x10 6 1.2x10 6

Amount 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00



### Regression details
R (Area Ratio) = B0 + B1 × A (Amount)
B0 = 1830 (intercept)
B1 = 10700 (gradient)
Correlation coefficient (r) = 0.999976
weighting used = none

Concentration (μg/mL) Peak area (μV.s) Calculated concentration (μg/mL) % Deviation

1 11084 0.86234 15.964

2.5 27399 2.38280 4.919

5 54741 4.93086 1.402

7.5 81035 7.38125 1.609

10 108380 9.92962 0.709

25 275199 25.47597 -1.868

50 542437 50.38067 -0.756

75 804128 74.76849 0.310

100 1073671 99.88802 0.112Study Number 8260178 Final Report

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### Figure 20 Purity and content of impurities anal ysis – chromatogram for acetonitrile

SampleName: Acetonitrile Date Acquired: 22-Mar-2012 16:20:39 GMT +00:00 Result Id: 303322.08924.16124.51027.63555.252AU

-1.40 -1.20 -1.00 -0.80 -0.60 -0.40 -0.20 0.00

Minutes

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.0 0



### Figure 21 Purity and content of impurities analys is – chromatogram for

SampleName: Date Acquired: 22-Mar-2012 20:23:38 GMT +00:00 Result Id: 30353.87320.90422.13824.15124.48927.79655.237AU

-1.40 -1.20 -1.00 -0.80 -0.60 -0.40 -0.20 0.00 0.20

Minutes

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.0 0Study Number 8260178 Final Report

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### Figure 22 Purity and content of impurities anal ysis – chromatogram for

SampleName: Date Acquired: 22-Mar-2012 21:44:35 GMT +00:00 Result Id: 303022.19624.20824.55027.79855.256AU

-1.40 -1.20 -1.00 -0.80 -0.60 -0.40 -0.20 0.00

Minutes

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.0 0



### Figure 23 Purity and content of impurities anal ysis – chromatogram for

SampleName: Date Acquired: 22-Mar-2012 23:05:33 GMT +00:00 Result Id: 303622.15924.16224.50227.79455.261AU

-1.40 -1.20 -1.00 -0.80 -0.60 -0.40 -0.20 0.00

Minutes

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.0 0Study Number 8260178 Final Report

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### Figure 24 Purity and content of impurities analys is – chromatogram for test substance

SampleName: ESTS 06/12 Date Acquired: 22-Ma r -2012 17:41:36 GMT +00:00 Result Id: 3027- 3.892- 20.23522.51524.33924.70527.84355.263AU

-1.50 -1.00 -0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Minutes 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.0 0Study Number 8260178 Final Report

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### APPENDICESStudy Number 8260178 Final Report

- 47 - 

### Appendix 1 Certificate of Analysis

PERFORME D UNDER GLP

Test Material

Grade Substance as Manufactured

Batch/Lot 12166

Date of Receipt 11 January 2012

Supplier

. Physical Appearance White crystalline powder

Recommended Storage Room temperature

Date of Analysis 22 March 2012

Purity 99.5% Material is considered stable and suitab le for use for one year, when it becomes subject to retest

Retest and expiry date 22 March 2013

Performing Laboratory Smithers Viscient (ESG) Ltd. Otley Road, Harrogate HG3 1PY, United Kingdom

Study Reference 8260178

Study Director Ben Foster

Quality Assurance Diane Raper

Date of Authorisation 21 December 2012

Certificate Reference 8260178-1009885-L01-D01Study Number 8260178 Final Report

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### Appendix 2 NMR Phas e ReportStudy Number 8260178 Final Report

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- 79 - 

### Appendix 3 Analytical Procedu re CLE (PC) 8260178-01DStudy Number 8260178 Final Report

- 80 - 

### SAFETY PRECAUTIONS
Operators should take the normal precaution of wearing gloves, laboratory coats and safety glasses when handling compound and matrix samples.
Safety assessments (Control of Substances Hazardous to Health, COSHH) have been made of those procedural steps involving preparation of solutions, reagents and analysis of matrix samples. Appropriate sa fety codes have been included in the text and are defined in the section titled General Handling Control Categories.

The hazards and risks of the substances haza rdous to health used in this method have been considered. Provided the method is accurately followed and the control measures specified in the method are used, there s hould be no foreseeable hazards to health.

INTRODUCTION

Concentrations of in solvent are determined by HPLC with UV detection at 305 nm (HPLC-UV), following dilution with mobile phase.

APPARATUS, MATERIALS, RE AGENTS AND SOLUTIONS
Apparatus and Glassware

• HPLC system: Waters 2695 series HPLC system

• Various laboratory glassware

• Various pipettes

• Short form pipettes

Equivalent equipment may be used.

Materials

• Acetonitrile Rathburn, HPLC grade

• Water Double distilled, in house

• Ammonium acetate Sigm a Aldrich, ACS grade

• Ammonium hydroxide Sigm a Aldrich, ACS grade

Equivalent or better grade r eagents/solvents may be used.

Reagents and Solutions [1a/b, 4d]

HPLC Mobile Phase A Dissolve ammonium acetate (1.5g) in double di stilled water (1000 mL ) and adjust to pH 9 with ammonium hydroxide.

HPLC Mobile Phase B HPLC grade acetonitrile.Study Number 8260178 Final Report

- 81 - 

### PROCEDURES

All work should be carried out under the mi nimum control categories listed under the safety precautions section. A dditional controls are listed w ith the individual steps of the procedure.

Standard Solution Preparation [1a/b, 4b/4d] Initial Weighing of Stock Solutions Prepare duplicate analytical standard solutio ns (A and B), corrected for purity, in a suitable solvent in volumetric flasks. The amount weighed must be at or slightly above the desired weight so that the solven t volume can be corrected to ensure that these stock solutions and their subseq uent dilutions are whole units, (eg 100.0 μg/mL).
Dilution into Calibration Range Dilute one standard (A) over the range of concentrations to be used for the instrument calibration in the chosen in jection solvent. Dilute th e second standard (B) to a concentration near the mid-point of this ca libration range, yet identical to a calibration point.

Standard Correlation Correlate the standard solutions by injecti ng each of the two standards (A and B), at a concentration near the mid-calibration poi nt, 5 times into the chosen analytical system. Ensure that the two solutions are injected alternately in the run sequence. The results for the correlation should be between 98 and 102 % of the mean. Then inject the calibration line to ensure that the standards meet the acceptance criteria.
Review of Results If the correlation is out of specification, either, repeat the injections, re-dilute or prepare two new stock standards and re peat the procedures in sections <<Initial Weighing of Stock Solutions>> to <<Review of Results>>

. If the calibration line is out of specification, either, repeat the inje ctions or re-dilute the standards and re-inj ect the calibration line.
If the acceptance criteria from section << Standard Correlation>> have been met, then the calibration solutions are acceptable fo r use. If required, fo rtification solutions for method validation will be made from the same stock standard, or its dilutions, from which the calibration line has been prepared.
New Calibration Line Preparation from Stock Solution A or B If a new calibration line is prepared, re peat the procedures in sections << Dilution into Calibration Range >> to << Review of Results >> above, correlating the new near midpoint standard against the old near mid-poi nt standard. Inject the new calibration line to check linearity. Review the data as in section << Review of results >>.Study Number 8260178 Final Report

- 82 - 

### New Standard Preparation Prepare a new analytical standard solution (C ) in a suitable solvent in a volumetric flask as described in section << Initial weighing of Stock Solutions>> . Dilute and correlate against an old near mid-point standard, as described in sections <<Dilution into Calibration Range>> to <<Review of Results>> above. Review the data and return to section <<Initial Weighing of Stock Solutions>> if the new standard is out of specification.

Calibration Standards [1a/b, 4b] Prepare appropriate calibration standards in HPLC grade aceton itrile to cover the range 1 to 100 μg/mL, with a lowest calibration level of 1.0 μg/mL. Calibration standards may be stored refr igerated for up to one month.
Sample Preparation Samples are diluted with HPLC grade acetonitr ile to bring the concentrations within the calibration range of 1 to 100 μg/mL. [1a/b, 4a]

Conditions for HPLC Analysis with UV Detection

Instrumentation: Waters 2695 HPLC system Column#: X-bridge RP18 3.5 μm, 150 x 4.6 mm Flow rate#: 1.0 mL/min Mobile phase A 0.02M ammonium acetate adjusted to pH9 Mobile phase B Acetonitrile (HPLC grade) Gradient Time (mins) A (%) B (%) 0 95 5 5 65 35 10 65 35 15 95 5 20 95 5 Column temperature#: 25 °C Injection volume#: 10 μL UV wavelength#: 305 nm Retention time: ca 5 minutes
Parameters marked # may not be modifi ed. Minor adjustments to the remaining parameters may be required in order to fully optimise the system.Study Number 8260178 Final Report

- 83 - 

### CALCULATION OF RESULTS
All peak measurements and calcu lations are performed on Empower TM 

### 2. From the measured peak area, where the calibration f it is linear as in this study, Empower uses the following formula to calculate the c oncentration of present in the sample.



### 1 23 Degree1st Constant - AreaPeak g/mL)(A UP UPxUP x = μ

Where:-
A (μg/mL) = Amount of Peak Area = Area of peak due to Constant = Y intercept on calibration graph 1st Degree = Slope of calibration graph UP1 = Sample amount UP2 = Final volume (mL) UP3 = Dilution

METHOD CRITERIA


### Analysis by HPLC will be considered successful only if the following criteria are met.

• At least 6 calibration standards will be used in the determination of each calibration line

• A correlation of coefficient (r) for each calibration line will be ≥ 0.99 or if using a 1/x weighing a coefficient of determination (r 2 

### ) for each calibration line will be ≥ 0.98

• All test samples will be within the appropriate calibration standards rangeStudy Number 8260178 Final Report

- 84 - 

### CATEGORY Main Division CONTROL Name and Specification 1

a b

c d GLOVES Disposable latex Disposable nitrile Rubber gloves Specific type for the job (see assessment giving details) 2

a b

c d

e PROTECTIVE CLOTHING Laboratory coat or equivalent Disposable overalls Oversleeves Overshoes Plastic apron 3

a b

c EYE/FACE PROTECTION Safety glasses to BS 2092/2 C or better Face shield to BS 2092/2 C or better Safety goggles to BS 2092/2 C or better 4

a b

c d

e f g ENGINEERING CONTROLS Open bench in ventilated area Fume cupboard to BS 7258 Laminar flow cabinet to BS 5295 Class 1 Re-circulating fume chamber Radioisotope lab Biohazard lab Glove box 5

a

b

c RESPIRATORY PROTEC TIVE EQUIPMENT Disposable filtering facemask (HSE approved), i - organic vapour ii - dust iii – combination organic vapour/dust MUST SPECIFY TYPE Powered respirators/helmets with safety visor to BS 2092/2 C or better (HSE approved) Respirator with specified canister (HSE approved) 6 SPECIFIC IMMUNISATION REQUIRED (GIVE DETAILS) 7 ALLERGIC PERSONS PROHIBIT ED (SPECIFY ALLERGY) 8 REFER TO MATERIAL SAFETY DATA SHEET 9 KNOWN OR SUSPECTED RE PRODUCTIVE HAZARD TO EITHER SEX (must specify details) 10 POISON – ensure antidote is availa ble and is within its expiry date (must specify details)Study Number 8260178 Final Report

- 85 - 

### Appendix 4 Analytical Procedu re CLE (PC) 8260178-02DStudy Number 8260178 Final Report

- 86 - 

### SAFETY PRECAUTIONS


### Operators should take the normal precaution of wearing gloves, laboratory coats and safety glasses when handling compound and matrix samples.
Safety assessments (Control of Substances Hazardous to Health, COSHH) have been made of those procedural steps involving preparation of solutions, reagents and analysis of matrix samples. Appropriate safety codes have been included in the text and are defined in the section titled General Handling Control Categories.

The hazards and risks of the substances haza rdous to health used in this method have been considered. Provided the method is accurately followed and the control measures specified in the method are correctly used, there should be no foreseeable hazards to health.

INTRODUCTION



### Concentrations of are determined by HPLC with UV detection at 205 nm (H PLC-UV), following dilution with HPLC gradient grade acetonitrile. This is a qualita tive method to determine the presence of impurities. Possible impurities might include
, and

.

APPARATUS, MATERIALS, RE AGENTS AND SOLUTIONS
Apparatus and Glassware



### • HPLC system: Waters 2695 series HPLC system

• Various laboratory glassware

• Various pipettes

• Short form pipettes

Equivalent equipment may be used

Materials



### • Acetonitrile Fisher, HPLC gradient grade

• Water Double distilled, in house

• Ammonium acetate Sigma Aldrich, ACS grade

• Ammonium hydroxide Sigm a Aldrich, ACS grade

• Sigm a Aldrich, ACS grade

• Sigm a Aldrich, ACS grade

• Sigm a Aldrich, ACS grade

Equivalent or better grade r eagents/solvents may be used.Study Number 8260178 Final Report

- 87 - 

### Reagents and Solutions [1a/b, 4d]

HPLC Mobile Phase A 

### Dissolve ammonium acetate (1.5g) in double distilled water (1000 mL) and adjust to pH 9 with ammonium hydroxide.

HPLC Mobile Phase B 

### HPLC grade acetonitrile.

PROCEDURES



### All work should be carried out under the mi nimum control categories listed under the safety precautions section. A dditional controls are listed with the individual steps of the procedure.
Sample Preparation 

### Samples are diluted with HPLC gradient grade acetonitrile to sufficiently high concentration to determine peaks not due to the main TA peak or those that might be present in the acetonitrile. A suitable c oncentration is 10,000 μg/mL or higher. Comparison with suitable concentrations of dissolved in HPLC gradient acetonitrile. [1a/b, 4a]

Conditions for HPLC Analysis with UV Detection



### Instrumentation: Waters 2695 HPLC system Column#: X-bridge 3.5 μm, 150 x 4.6 mm Flow rate#: 1.0 mL/min Gradient Time (mins) A (%) B (%) 0 95 5 5 95 5 60 5 95 65 5 95 70 95 5 80 95 5 Column temperature#: 25 °C Injection volume#: 10 μL UV wavelength#: 205 nm Retention time: ca 20 minutes for the main TA peak

Parameters marked # may not be modifi ed. Minor adjustments to the remaining parameters may be required in order to fully optimise the system.Study Number 8260178 Final Report

- 88 - 

### CALCULATION OF RESULTS


### All peak measurements and calcu lations are performed on Empower TM 

### 2. From the measured peak area, where the calibration fit is linear as in this study, Empower uses the following formula to calculate the c oncentration of present in the sample.

1 23 Degree1st Constant - AreaPeak g/mL)(A UP UPxUP x = μ

Where:-
A (μg/mL) = Amount of Peak Area = Area of peak due to Constant = Y intercept on calibration graph 1st Degree = Slope of calibration graph UP1 = Sample amount UP2 = Final volume (mL) UP3 = DilutionStudy Number 8260178 Final Report

- 89 - 

### METHOD CRITERIA


### Analysis by HPLC will be considered successful only if the following criteria are met.

• At least 6 calibration standards will be used in the determination of each calibration line

• A correlation of coefficient (r) for each calibration line will be ≥ 0.99 or if using a 1/x weighing a coefficient of determination (r 2 

### ) for each calibration line will be ≥ 0.98

• All test samples will be within the appropriate calibration standards rangeStudy Number 8260178 Final Report

- 90 - 

### GENERAL HANDLING CONTROL CATEGORIES



### CATEGORY Main Division CONTROL Name and Specification 1

a b

c d GLOVES Disposable latex Disposable nitrile Rubber gloves Specific type for the job (see assessment giving details) 2

a b

c d

e PROTECTIVE CLOTHING Laboratory coat or equivalent Disposable overalls Oversleeves Overshoes Plastic apron 3

a b

c EYE/FACE PROTECTION Safety glasses to BS 2092/2 C or better Face shield to BS 2092/2 C or better Safety goggles to BS 2092/2 C or better 4

a b

c d

e f g ENGINEERING CONTROLS Open bench in ventilated area Fume cupboard to BS 7258 Laminar flow cabinet to BS 5295 Class 1 Re-circulating fume chamber Radioisotope lab Biohazard lab Glove box 5

a

b

c RESPIRATORY PROTEC TIVE EQUIPMENT Disposable filtering facemask (HSE approved), i - organic vapour ii - dust iii – combination organic vapour/dust MUST SPECIFY TYPE Powered respirators/helmets with safety visor to BS 2092/2 C or better (HSE approved) Respirator with specified canister (HSE approved) 6 SPECIFIC IMMUNISATION REQUIRED (GIVE DETAILS) 7 ALLERGIC PERSONS PROHIBIT ED (SPECIFY ALLERGY) 8 REFER TO MATERIAL SAFETY DATA SHEET 9 KNOWN OR SUSPECTED RE PRODUCTIVE HAZARD TO EITHER SEX (must specify details) 10 POISON – ensure antidote is availa ble and is within its expiry date (must specify details)