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 90 



Study Number 8260178 

Final Report 

- 5 - 

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 elements 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 Smith 
Study Number 8260178 

Final Report 

- 6 - 

ARCHIVE STATEMENT 

 Determination of the IR, 

UV/Vis, Mass and NMR spectra, Purity Assessment and Identification of 

Impurities 

All primary data, or authenticated copies thereof, specimens or samples will be 

retained in the Covance Laboratories Limited archives, on behalf of Smithers Viscient 

(ESG) Limited, for ten years after issue of the final report. At this time, the Sponsor 

will be contacted to determine whether the data should be returned, retained or 

destroyed on their behalf. Sponsors will be notified of the financial implications of 

each of these options at that 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 commencement of NMR 

analysis and finished on 12 April 2012 with the assignment of absorption peaks for 

the determination of IR spectra. Study completion 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 

- 7 - 

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 Absorption Spectroscopy...................................................................................................11

Ultraviolet-Visible Spectroscopy ....................................................................................................12

Mass Spectrometry..........................................................................................................................12

Nuclear Magnetic Resonance 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 Substance for Impurities.......................................................................................14

RESULTS ..............................................................................................................................................15

Infrared Absorption Spectroscopy...................................................................................................15

Ultraviolet-Visible Spectroscopy ....................................................................................................15

Mass Spectrometry..........................................................................................................................16

Nuclear Magnetic Resonance Spectroscopy ...................................................................................16

Analytical Procedure 16

Analytical Procedure for Identification and Analysis of Impurities................................................17

Analysis of Test Substance Purity...................................................................................................17

Analysis of Test Substance for Impurities.......................................................................................17

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

TABLES.................................................................................................................................................20

Table 1 Infrared spectral details .....................................................................................................21

Table 2 Ultraviolet-visible spectral characteristics, 

....................................................................................................................................22

Table 3 Precision results ................................................................................................................23

Table 4 Accuracy (recovery) results ..............................................................................................24

Table 5 Determination of test substance purity and impurity content............................................25
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FIGURES ...............................................................................................................................................26

Figure 1 Structure of ..................................27

Figure 2 Infrared spectrum 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 spectra for ...........................................................34

Figure 9 Proposed fragmentation pathway for ...............................................35

Figure 10 Negative ion mass spectra for ............................................................36

Figure 11 Proposed fragmentation pathway for .................................................37

Figure 12 Analytical procedure – chromatogram for acetonitrile ..................................................38

Figure 13 Analytical procedure – chromatogram for 1.0 μg/mL calibration standard...................38

Figure 14 Analytical procedure – chromatogram for 10 μg/mL calibration standard....................39

Figure 15 Analytical procedure – chromatogram for 100 μg/mL calibration standard..................39

Figure 16 Analytical procedure – chromatogram for 2 μg/mL recovery sample ...........................40

Figure 17 Analytical procedure – chromatogram for 50 μg/mL recovery sample .........................40

Figure 18 Analytical procedure – chromatogram for 95 μg/mL recovery sample .........................41

Figure 19 Analytical procedure - calibration line...........................................................................42

Figure 20 Purity and content of impurities analysis – chromatogram 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 impurities analysis – chromatogram for .....................44

Figure 24 Purity and content of impurities analysis – chromatogram for test substance ...............45

APPENDICES........................................................................................................................................46

Appendix 1 Certificate of Analysis................................................................................................47

Appendix 2 NMR Phase Report.....................................................................................................48

Appendix 3 Analytical Procedure CLE (PC) 8260178-01D ..........................................................79

Appendix 4 Analytical Procedure CLE (PC) 8260178-02D ..........................................................85
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SUMMARY 

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) 

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 maxima were 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 neutralisation 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 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 

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 system to 

was linear, over the range 1 to 

100 μg/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 

The method was therefore considered specific for to 
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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% 

 
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INTRODUCTION 

The test substance, was 

allocated the code ESTS 06/12 for reference throughout the study. 

Name: 

Chemical Structure: 

CAS Number: 

Chemical 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 methods for purity 

assessment and for identification of impurities for the test substance, . 

PROCEDURES 

Protocol Adherence 

The study was performed in accordance with the protocol and three amendments. 

Infrared Absorption Spectroscopy 

Method 

The test substance was combined with potassium bromide, ground to a very fine 

powder, and formed into an IR-transparent disc under hydraulic pressure. 

 

1

 Determined within this study 
Study Number 8260178 

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The spectrum was recorded on a Perkin Elmer Spectrum One instrument against a 

blank potassium bromide disc at a temperature 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 experimental procedures followed those described in OECD Guideline 101. 

Method 

Methylene Blue solution was used to verify the correct operation of the spectrometer, 

with a spectrum recorded between 190 and 750 nm in 10 mm pathlength in a far-UV 

quartz cell. The absorbance wavelengths obtained were compared to those in the 

literature and the spectrometer was considered to be operating 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 988, 1016 and 1037 μg/mL in methanol (neutral), 

in methanol containing 10% 1 mol/L hydrochloric acid (acidic) and in methanol 

containing 10% 0.999 mol/L sodium hydroxide 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 strips). 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 instrument 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 solution 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: ThermoFisher LTQ Orbitrap hybrid mass spectrometer 

Ionisation Mode: Positive ion electrospray 

Scan Range: MS: 95-600 m/z, MS2: data dependant 

Source Voltage: 4.5 kV 

Capillary Temperature: 280°C 

Resolution: MS: 60000, MS2 7500 
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LC/MS conditions for 

Instrumentation: ThermoFisher LTQ Orbitrap hybrid mass spectrometer 

Ionisation Mode: Negative ion electrospray 

Scan Range: MS: 115-400 m/z, MS2: data dependant 

Source Voltage: 3.5 kV 

Capillary Temperature: 280°C 

Resolution: MS: 60000, MS2

 7500 

Nuclear Magnetic Resonance Spectroscopy 

Method 

Full details of the procedure employed are presented 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 chromatography with ultra-violet detection 

(HPLC-UV), following dilution with acetonitrile to obtain an analytical solution. 

The full procedure can be found in Appendix 3. 

The method was assessed against the criteria detailed in the following sections: 

Specificity 

Test solutions were prepared at appropriate concentration levels along with a blank 

acetonitrile sample 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 the concentration range (nine different 

concentrations) was determined by least squares linear regression. 

Precision (Repeatability) 

For procedure 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 concentrations of 2 μg/mL, 50 μg/mL and 95 μg/mL. 

The prepared solutions were diluted, as detailed 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 analysis of prepared sample solutions at each of three concentrations. 

Solutions were prepared at concentrations of 2 μg/mL, 50 μg/mL and 95 μg/mL. 
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Each of the prepared solutions was diluted for HPLC-UV analysis, as detailed in the 

analytical procedure, to fall within the calibration range. The mean recovery, and 

RSD (relative standard deviation) were determined for each set of preparations. 

Analytical Procedure for identification and analysis of impurities and 

determination of purity 

The procedure CLE (PC) 8260178-02D for determining impurities in 

 was developed in this study. 

The method used high performance liquid chromatography with ultra-violet detection 

(HPLC-UV), following dilution with acetonitrile to obtain an analytical solution. 

The full procedure can be found in Appendix 4. 

No validation was carried out on this method due to the fact that no reference material 

was available for the test substance and the purity was calculated using percent peak 

area values. This is a deviation from the protocol 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 procedure 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 

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RESULTS 

Infrared Absorption Spectroscopy 

Table 1 lists the major peaks and their tentative 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 6th (2008) 

edition, McGraw-Hill. 

The peaks are characteristic of the test substance, 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 

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 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 

C26H36BNO6, and the molecular weight used to calculate the molar extinction 

coefficients (absorbance of a 1 cm path length of a solution containing 1 mole/L of 

substance) was . The tested molar concentrations were therefore: 

Neutral conditions - for the 19.76 and 9.88 μg/mL 

solutions, respectively. 

Acidic conditions - for the 20.32 and 10.16 μg/mL 

solutions, respectively. 

Basic conditions - for the 20.74 μg/mL solution. 

The test results 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 unadjusted 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 under basic conditions is considered to 

result from an irreversible change such as hydrolysis or degradation rather than a 
Study Number 8260178 

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reversible conformational change, 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 electrospray for analysis of the 

Tri-n-butylammonia 

Full spectrum MS demonstrated a base peak positive ion of m/z which was 

consistent with the structure of 

MS2

 fragmentation of m/z yielded a product ion spectrum containing ions of 

m/z

The mass spectra are shown in Figure 8. 

A proposed fragmentation pathway is shown in Figure 9. 

Full spectrum MS demonstrated a base peak negative ion of m/z , which was 

consistent with the structure of 

MS2

 fragmentation of m/z yielded a product ion spectrum containing an ion 

of m/z 

The mass spectra are shown in Figure 10. 

A proposed fragmentation 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 methods were assessed against the criteria detailed in the following sections: 

Specificity 

All solutions were free of any components that interfered with the analysis of the test 

substance. The method was therefore considered specific for 

. Example chromatograms of acetonitrile and test 

substance solutions are presented in Figure 12 to Figure 18. 
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Linearity 

Response of the HPLC-UV system to 

 was linear, over the range 1 to 100 μg/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 method, expressed as the relative standard deviation (RSD) 

measurements for obtained from 

solutions in acetonitrile, were within the acceptance criteria specified in 

SANCO/3030/99 (rev 4) of <9.34% for the 2 μg/mL2

 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 precision data are shown in Table 3. Example chromatograms are 

presented in Figure 16 to Figure 18. 

Accuracy (Recovery) 

Mean recoveries 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/mL recovery levels. The accuracy data are shown in Table 4, 

respectively. Example chromatograms are presented in Figure 16 to Figure 18. 

Analytical Procedure for Identification 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 interfering peaks from the acetonitrile blank. 

Analysis of Test Substance Purity 

Analysis of the test substance using the conditions stated in procedure CLE (PC) 

8260178-02D produced two chromatographic 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. Chromatograms 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 calculation. 3

 Recovery acceptance ranges were taken from SANCO guideline 3030/99 (rev 4) for the nominal spiked accuracy 

concentrations used. See Table 4 for details. 
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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, indicating no change in conformation. 

In basic solution three absorbance maxima were 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 

neutralisation 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 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 system to 

 was linear, over the range 1 to 

100 μg/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 

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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% 

 
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TABLES 
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Table 1 

 Infrared spectral details 
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Table 2 

 Ultraviolet-visible spectral characteristics, 

Conditions Maximum absorbance wavelength (nm) Extinction coefficient (ε) 

 
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Table 3 

 Precision 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 (revision 4) are based on the modified Horwitz equation: 

%RSDR = 2(1-0.5 x log C)

Where: 

RSDR = 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: 

%RSDr = %RSDR x 0.67 

For acceptance of results the precision values must be below the corresponding RSDr value. 

For QC 2 RSDr = 2(1-0.5 x log 0.0000025) x 0.67 = 9.34 

For QC 50 precision RSDr = 2(1-0.5 x log 0.000064) x 0.67 = 5.73 

For QC 95 precision RSDr = 2(1-0.5 x log 0.00012) x 0.67 = 5.21 
Study Number 8260178 

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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 ingredient content of <0.01% 

90 – 110% for a nominal active ingredient content of 0.01% to 0.1%
Study Number 8260178 

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Table 5 

 Determination of test substance 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 0.5 

 
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FIGURES 
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Figure 1 

 Structure of 
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Figure 4 

 UV / Visible spectrum for neutral solution in methanol 
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Figure 5 

 UV / Visible spectrum for acidic solution in methanol 
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Figure 6 

 UV / Visible spectrum for basic solution in methanol 
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Figure 7 

 UV / Visible spectrum for basic solution in methanol after neutralisation 
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Figure 8 
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Figure 10 
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Figure 12 

 Analytical procedure – chromatogram for acetonitrile 

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

AU

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 20.00

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 - 7.171

AU

-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 20.00
Study Number 8260178 

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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 - 7.166

AU

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 20.00

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 - 7.157

AU

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 20.00

 
Study Number 8260178 

Final Report 

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Figure 16 

 Analytical procedure – chromatogram for 2 μg/mL recovery sample 

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

AU

-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 20.00

Figure 17 

 Analytical procedure – chromatogram for 50 μg/mL recovery sample 

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

AU

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 20.00
Study Number 8260178 

Final Report 

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Figure 18 

 Analytical procedure – chromatogram for 95 μg/mL recovery sample 

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

AU

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 20.00
Study Number 8260178 

Final Report 

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Figure 19 

 Analytical procedure - calibration line 

A rea

-2.0x105

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

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 0.112 
Study Number 8260178 

Final Report 

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Figure 20 

 Purity and content of impurities analysis – chromatogram for acetonitrile 

SampleName: Acetonitrile Date Acquired: 22-Mar-2012 16:20:39 GMT +00:00 Result Id: 3033 22.089 24.161 24.510 27.635 55.252

AU

-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 80.00

Figure 21 

 Purity and content of impurities analysis – chromatogram for 

SampleName: Date Acquired: 22-Mar-2012 20:23:38 GMT +00:00 Result Id: 3035 

3.873

20.904 22.138

24.151 24.489

27.796

55.237

AU

-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 80.00
Study Number 8260178 

Final Report 

- 44 - 

Figure 22 

 Purity and content of impurities analysis – chromatogram for 

SampleName: Date Acquired: 22-Mar-2012 21:44:35 GMT +00:00 Result Id: 3030 22.196 24.208 24.550 27.798 55.256

AU

-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 80.00

Figure 23 

 Purity and content of impurities analysis – chromatogram for 

SampleName: Date Acquired: 22-Mar-2012 23:05:33 GMT +00:00 Result Id: 3036 22.159 24.162 24.502 27.794 55.261

AU

-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 80.00
Study Number 8260178 

Final Report 

- 45 - 

Figure 24 

 Purity and content of impurities analysis – chromatogram for test substance 

SampleName: ESTS 06/12 Date Acquired: 22-Mar-2012 17:41:36 GMT +00:00 Result Id: 3027 - 3.892 - 20.235 22.515 24.339 24.705 27.843 55.263

AU

-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 80.00
Study Number 8260178 

Final Report 

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APPENDICES 
Study Number 8260178 

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Appendix 1 

 Certificate of Analysis 

PERFORMED 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 suitable 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-D01 

 
Study Number 8260178 

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Appendix 2 

 NMR Phase Report 
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Appendix 3 

 Analytical Procedure CLE (PC) 8260178-01D 
Study 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 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 hazardous 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 should 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, REAGENTS 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 Sigma Aldrich, ACS grade 

• Ammonium hydroxide Sigma Aldrich, ACS grade 

Equivalent or better grade reagents/solvents may be used. 

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. 
Study Number 8260178 

Final Report 

- 81 - 

PROCEDURES 

All work should be carried out under the minimum control categories listed under the 

safety precautions section. Additional controls are listed with the individual steps of 

the procedure. 

Standard Solution Preparation [1a/b, 4b/4d] 

Initial Weighing of Stock Solutions 

Prepare duplicate analytical standard solutions (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 solvent volume can be corrected to ensure that 

these stock solutions and their subsequent 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 injection solvent. Dilute the second standard (B) to a 

concentration near the mid-point of this calibration range, yet identical to a calibration 

point. 

Standard Correlation 

Correlate the standard solutions by injecting each of the two standards (A and B), at a 

concentration near the mid-calibration point, 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 repeat the procedures in sections <<Initial 

Weighing of Stock Solutions>> to <<Review of Results>>. 

If the calibration line is out of specification, either, repeat the injections or re-dilute 

the standards and re-inject the calibration line. 

If the acceptance criteria from section <<Standard Correlation>> have been met, 

then the calibration solutions are acceptable for use. If required, fortification 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, repeat the procedures in sections <<Dilution into 

Calibration Range>> to <<Review of Results>> above, correlating the new near midpoint standard against the old near mid-point standard. Inject the new calibration line 

to check linearity. Review the data as in section <<Review of results>>. 
Study Number 8260178 

Final Report 

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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 acetonitrile to cover the 

range 1 to 100 μg/mL, with a lowest calibration level of 1.0 μg/mL. Calibration 

standards may be stored refrigerated for up to one month. 

Sample Preparation 

Samples are diluted with HPLC grade acetonitrile 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 modified. 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 calculations are performed on EmpowerTM2. From the 

measured peak area, where the calibration fit is linear as in this study, Empower uses 

the following formula to calculate the concentration of present in the 

sample. 

1

23

Degree1st 

Constant - AreaPeak g/mL)(A UP

UPxUP

μ = x 

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 (r2

) for each calibration line will be 

≥ 0.98 

• All test samples will be within the appropriate calibration standards range 
Study 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 PROTECTIVE 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 PROHIBITED (SPECIFY ALLERGY) 

8 REFER TO MATERIAL SAFETY DATA SHEET 

9 KNOWN OR SUSPECTED REPRODUCTIVE HAZARD TO 

EITHER SEX (must specify details) 

10 POISON – ensure antidote is available and is within its expiry 

date (must specify details) 
Study Number 8260178 

Final Report 

- 85 - 

Appendix 4 

 Analytical Procedure CLE (PC) 8260178-02D 
Study 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 hazardous 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 (HPLC-UV), following dilution with HPLC 

gradient grade acetonitrile. This is a qualitative method to determine the presence of 

impurities. Possible impurities might include , and 

. 

APPARATUS, MATERIALS, REAGENTS 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 Sigma Aldrich, ACS grade 

• Sigma Aldrich, ACS grade 

• Sigma Aldrich, ACS grade 

• Sigma Aldrich, ACS grade 

Equivalent or better grade reagents/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 minimum control categories listed under the 

safety precautions section. Additional 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 concentration 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 modified. 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 calculations are performed on EmpowerTM2. From the 

measured peak area, where the calibration fit is linear as in this study, Empower uses 

the following formula to calculate the concentration of present in the 

sample. 

1

23

Degree1st 

Constant - AreaPeak g/mL)(A UP

UPxUP

μ = x 

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 
Study 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 (r2

) for each calibration line will be 

≥ 0.98 

• All test samples will be within the appropriate calibration standards range 
Study 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 PROTECTIVE 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 PROHIBITED (SPECIFY ALLERGY) 

8 REFER TO MATERIAL SAFETY DATA SHEET 

9 KNOWN OR SUSPECTED REPRODUCTIVE HAZARD TO 

EITHER SEX (must specify details) 

10 POISON – ensure antidote is available and is within its expiry 

date (must specify details)