### JRF Study Number: 240-2-11-5551 (Final Report) Page 2 of 52


### STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any informati on contained in this study on the basis of its falling within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C).

Sponsor Tiarco Chemical 1300 Tiarco Drive Dalton, GA 30720

Signature ___________________________ Sponsor/Submitter

Date ____________________________

### JRF Study Number: 240-2-11-5551 (Final Report) Page 6 of 52


### CONTENTS PAGE N° TITLE PAGE .................................................................................................................... ....................... 1 STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS ............................................................ 2 STATEMENT OF GLP COMPLIANCE ................................................................................................ 3 STATEMENT OF COMPLIANCE WITH GOOD LABORATORY PRACTICE ................................ 4 STATEMENT OF QUALITY ASSURANCE ........................................................................................ 5 CONTENTS ...................................................................................................................... ...................... 6 PERSONNEL INVOLVED IN THE STUDY ........................................................................................ 8 SUMMARY ....................................................................................................................... ...................... 9 Infrared Analysis ............................................................................................................ ................... 9 LC-Mass An alysis ............................................................................................................. .............. 10 NMR Analysis ................................................................................................................. ................ 10 Active Ingredient Content .................................................................................................... ........... 10 1. INTRODUCTION ............................................................................................................... ............ 11 1.1 Study Objectives ......................................................................................................... ........... 11 1.2 Test Facility and Study Period ........................................................................................... ... 11 1.3 Regulatory Guidelines .................................................................................................... ....... 11 1.4 Archives.................................................................................................................. ............... 11 2. EXPERIMENTAL PROCEDURE ................................................................................................. 12 2.1 Test Item ................................................................................................................ ................ 12 2.2 Referen ce Standard ....................................................................................................... ......... 12 2.3 Equipment/Instruments .................................................................................................... ..... 13 2.4 Solvents, Chemicals and Reagents ........................................................................................ 13 2.5 Intr oduction ............................................................................................................. .............. 13 2.6 Interpretation ........................................................................................................... .............. 13 2.7 Methodology for IR Spectral Analysis .................................................................................. 14 2.8 Procedure for the Determination of Active Ingredient Content ............................................ 14 2.8.1 Preparation of Sodium 3-Nitr obenzene Sulfonate Reference Standard Stock Solution .......................................................................................................... 14 2.8.2 Preparation of Reference St andard Solutions for Linearity ...................................... 14 2.8.3 Preparation of Reference Standard Solution for Active Ingredient Analysis ........... 15

### JRF Study Number: 240-2-11-5551 (Final Report) Page 7 of 52 

### CONTENTS (Continued) PAGE N° 2.8.4 Preparation of Sample Solutions .............................................................................. 15 2.8.5 Instrumental Parameters ........................................................................................... 15 2.9 Procedure for LC-Mass Spectral Analysis ............................................................................ 15 2.9.1 Preparation of Sodium 3-nitrobenzen es sulfonate Reference Standard and Test Item Solutions........................................................................................................... 15 2.9.2 LC Mass Parameters ................................................................................................. 16 2.10 Procedure for NMR Analysis .............................................................................................. .. 16 2.10.1 Preparation of Sodium 3-nitrobe nzenes sulfonate Reference Standard and Test Item Solution .................................................................................................... 16 3. RESULTS .................................................................................................................... ................... 17 3.1 Infrare d Spectra ......................................................................................................... ............ 17 3.2 Mass An alysis ............................................................................................................ ............ 17 3.3 NMR An alysis ............................................................................................................. .......... 17 3.4 Active Ingredient Content ................................................................................................ ..... 17 4. CONCLUSION ................................................................................................................. .............. 18 5. REFERENCES ................................................................................................................. ............... 19 TABLES 1. Linearity Data of Sodium 3-Nitrobe nzene Sulfonate Reference Standard ............................ 20 2. Calculation of Active Ingredient Content .............................................................................. 21 APPENDICES 1. IR, Mass NMR Spectra an d HPLC Chromatograms ............................................................. 22 2. Signed Study Plan and Study Plan Amendment .................................................................... 35 3. Certificate of Analysis of Sodium 3- Nitrobenzenes Sulfonate Technical Sample ................ 49 4. Certificate of Analysis of Sodium 3-N itrobenzenes Sulfonate Refe rence Standard ............. 50 5. GLP Endorsement of Compliance ......................................................................................... 51

### JRF Study Number: 240-2-11-5551 (Final Report) Page 9 of 52


### SUMMARY

This study was conducted to perform the characteriza tion of sodium 3-nitrobenzene sulfonate. The test item was supplied by Tiarco Chemical, Dalton, GA 30720. The method was followed as per the guidelines of the SANCO/ 3030/99 re v. 4 AND U.S. EPA OPPTS 830.1800. IR and Mass analysis were carried out following th e Standard Operating Procedures of Jai Research Foundation. The IR and NMR spectral data were interpretated usi ng standard references William D. H. and Fleming I. (1988) and Robert M. S., Francis X. W (1997).

The structure of sodium 3-nitrobenzene sulfonate t est item was confirmed by comparing with sodium 3- nitrobenzene sulfonate reference standard using FT-IR based on the presence of the characteristic absorption bands due to various functional groups; using LC-MS based on molecular mass and retention time and using NMR based on the chemical shift and num bers of proton present in test item and standard. The active ingredient analysis was carried out usi ng validated analytical method (JRF Study Number: 228-2-13-5312)
Infrared Analysis The structures of sodium 3-nitrobenzene sulfonate refe rence standard and test item were confirmed based on the presence of the characteristic absorption bands due to various functional groups by infrared analysis as described below: Functional groups Reference Standard Sample - CH stretching 3106.76, 2936.09 a nd 2873.42 3102.9, 2935.13 and 2873.42 - CH bending 1467.56 1468.53 -C=C stretching in benzene ring 1611.23 1610.27

-NO 2 stretching 1536.99 and 1351.86 1536.02 and 1351.86

-SO 3 H stretching 1190.83, 1051.98 and 647.965 1192.76, 1047.16 and 670.142 -adjacent H- atom 764.637 764.637

### JRF Study Number: 240-2-11-5551 (Final Report) Page 10 of 52 LC-Mass Analysis Identification of sodium 3-nitrobenzene sulfonate refe rence standard and test item using LC-MS, based on
molecular mass and retention time is summarized below: Sample Retention Time (min) Mass (m/z)
Reference Standard 3.999 202.2
Test Item 4.018 202.2
NMR Analysis Identification of sodium 3-nitrobenzene sulfonate re ference standard and test item using NMR, based on chemical shift and numbers of proton present are summarised below:

Molecular Structure:

SO 3

NO 2 H

H H

H Na + -

1

2 3 4

Identification No of Protons Multiplicity Chemical Shift ( δ ) Integration


### Standard Sample Standard Sample

1 

### 1 Triplet 7.724 – 7.764 7.723 – 7.764 1.09 1.09

2 

### 1 Doublet 8.216 –8.242 8.216 –8.242 1.06 1.06

3 

### 1 Doublet 8.327- 8.369 8.327- 8.369 1.11 1.18

4 

### 1 Singlet 8.666 – 8.675 8.665 – 8.675 1.00 1.00



### Active Ingredient Content Active Ingredient Content of sodium 3-nitrobenzene sulfonate was carried out using validate analytical method [Reference JRF Study Number: 228-2-13-5312]. The mean active Ingredient Content of s odium 3-nitrobenzene sulfonate was 96.88%w/w

### JRF Study Number: 240-2-11-5551 (Final Report) Page 11 of 52


### 1. INTRODUCTION 1.1 Study Objectives This study was conducted to perform the charact erization of sodium 3-nitrobenzene sulfonate (CAS # 127-68-4, US EPA HPV 3). The study was conducted in compliance with the OECD Principles of GLP (1998).
1.2 Test Facility and Study Period This study was performed at the Department of Chemistry, Jai Research Foundation, Valvada – 396 108, Dist. Valsad, Gujarat, India. Study Initiation : October 12, 2012 Experiment Start : October 15, 2012 Experiment Completion : October 15, 2012 Study Completion : December 05, 2012 1.3 Regulatory Guidelines European Commission: Directorate General H ealth and Consumer Protection 2000: SANCO/ 3030/99 rev. 4 “Technical and Material Prepara tions: Guidance for generating and reporting methods of analysis in support of pre- and pos t-registration data requirements for Annex II (Part A Section 4) and Annex III (part A, Section 5) of Directive 91/414/EEC” as amended by European Commission Directive 94/37/EC and 96/46/EC. U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). United States Environmental Protection Agency, Product Properties Test Guidelines (August 1996).

1.4 Archives All original raw data including any storag e medium for electronically recorded data, documentation, IR spectra, mass spectra, NMR sp ectra, chromatograms, the signed study plan, the study plan amendment, the draft report, a copy of final report and the representative sample of
the test item will be retained in the GLP Arch ives at Jai Research Foundation for a period of ten years. At the end of this period, the sponsor's instructions will be sought to either extend the archiving period or return the archived material to the Sponsor or for the material to be disposed
of.

### JRF Study Number: 240-2-11-5551 (Final Report) Page 12 of 52

SO 3

NO 2 Na + - 

### 2. EXPERIMENTAL PROCEDURE 2.1 Test Item Details of the test item provided by the Sponsor are:
Test Item Name IUPAC Name C.A. Name CAS N° Batch N° Analysed Purity Manufacture by Supplied by Date of Manufacture Date of Expiry Appearance Sodium 3-Nitrobenzene Sulfonate Benzenesulfonic acid, 3-nitro, sodium salt (1:1) Benzenesulfonic acid, 3-nitro, sodium salt (1:1) 127-68-4 50623 96.9% (Refer Certificate of Analysis in Appendix 3) Tiarco Chemical Tiarco Chemical September 08, 2011 September 08, 2013 Yellow to tan granular Storage Condition (at JRF) As per the instruction received from the Sponsor on storage of the test substance, the test item was stored in its original container as supplied by the Sponsor at room temperature in the Test Item Control Office (TICO). The stability of the test item in storage was the responsibility of the Sponsor.

2.2 Reference Standard Reference Standard IUPAC Name C.A. Name Molecular Weight Molecular Formula CAS N° Lot N° Date of Receipt Date of Expiry Purity Source JRF Entry Number Storage Condition Sodium 3-Nitrobenzenes Sulfonate Benzenesulfonic acid, 3-nitro-, sodium salt (1:1) Benzenesulfonic acid, 3-nitro-, sodium salt (1:1) 225.15 O 2 

### NC 6 

### H 4 

### SO 3 

### Na [127-68-4] 07315KNV August 07, 2012 August 06, 2017 (as per JRF SOP-152) 98.0% [Refer Appendix 4] Sigma-Aldrich, USA CPC-54 Room Temperature
Source: Molecular structure was obtained from Internet.

### JRF Study Number: 240-2-11-5551 (Final Report) Page 13 of 52 2.3 Equipment/Instruments
Sr. N° Name Model Manufacturer 1 Balance GR-202 Adair Dutt 2 FT-IR Spectrometer FT IR-4100 Jasco

3 LC-MS Agilent 1100 series, binary pump with Agilent VWD/MSD Agilent

4 NMR 300 MHz Bruker 5 HPLC LC 2010 CHT Simadzu

2.4 Solvents, Chemicals and Reagents Sr. N° Name Grade Supplier
1 Water Milli- Q Milli pore Milli Q water purification system
2 Acetonirile HPLC s.d fine chem. ltd 3 Potassium bromide (KBr) IR Merck 4 Formic Acid HPLC Merck 5 Deutorated methanol NMR Qualigens

2.5 Introduction Sodium 3-nitrobenzenes sulfonate reference sta ndard and test item were analyzed by FT-IR Spectrometer, LC-MSD, NMR and HPLC (for A.I C ontent). The identification with FT-IR based on the presence of the characteristic absorption ba nds due to various functional groups; using LCMS based on molecular mass and retention time and using NMR based on the chemical shift and numbers of proton present in test item and standard. The active ingredient analysis was carried out using validated analytical method using HPLC.

2.6 Interpretation The spectral data was interpreted using the standa rd references viz., William D. H. and Fleming I. (1988) and Robert M. Silverstein, Francis X. Webster, 1997.

### JRF Study Number: 240-2-11-5551 (Final Report) Page 14 of 52 2.7 Methodology for IR Spectral Analysis Approximately 2.0 mg of 

### reference standard and test item was mixed with 100 mg (approx.) dried potassium bromide separately. The mixture was finely ground using pestle and mortar and the mixture was placed in DRA sample holder of sample compartment. Background spectrum was recorded before analyzing sample by using blank KBr. All samples and background spectra were r ecorded using the following parameters: Instrument : FT-IR Spectrometer Wave Number Range : 400-4000 cm -1


### Resolution : 4.0 cm -1



### 2.8 Procedure for the Determination of Active Ingredient Content 2.8.1 Preparation of Sodium 3-Nitrobenzene Sulfonate Reference Standard Stock Solution Weight (mg) of Reference Standard Final Volume (mL) Volume made using Purity (%) Obtained Concentration (mg/L) Identification of Reference Standard Stock Solution 25.47 25 Milli-Q Water 98.0 998.42 A



### 2.8.2 Preparation of Reference Standard Solutions for Linearity Reference Standard Stock Solution Solution Taken (mL) Final Volume (mL) Volume made using Obtained Concentration (mg/L) Identification of Reference Standard Solution

A 0.25

10 Milli-Q Water 24.96 B 0.50 49.92 C 0.75 74.88 D 1.00 99.84 E 1.25 124.8 F 1.50 149.76 G 

### The reference standard solutions [B, C, D, E, F and G] were injected onto HPLC in duplicate using the parameters in accordance with section 2.8.5 and the mean peak area was plotted against concentrations (mg/L). The correlation coefficient (r), intercept with y-axis (a) and slope of the line (b) were calculated and the regression equation Y = bX + a was established.

### JRF Study Number: 240-2-11-5551 (Final Report) Page 15 of 52 2.8.3 Preparation of Reference Standard Solution for Active Ingredient Analysis The reference standard solution (E), concentration 99.84 mg/L , prepared for linearity was used for determination of active i ngredient content in the sample.

2.8.4 Preparation of Sample Solutions

Replication Weight of Standard/Test

Item (mg) Volume Made using (mL) Identification of Stock Solution Volume taken Final Volume (mL)

I 24.20 25 R 1 1.0 10

II 24.34 25 R 2 1.0 10 

### Above solutions were prepared in Milli-Q Water

2.8.5 Instrumental Parameters The above prepared standard solution, sample solutions and Milli-Q water were injected onto High Performance Liquid Chromatograph (HPLC) using validated parameters(JRF Study Number 228-2-13-5312). Instrument : HPLC Column : C-18 [250 mm x 4.6 mm (i.d.) x 5 μm particle size] Wave length : 260 nm Flow rate : 1.0 mL/minute Injection volume : 20 μL Mobile Phase : Acetonitrile (70): 0.1 % formic acid in water (30) v/v

2.9 Procedure for LC-Mass Spectral Analysis 2.9.1 Preparation of Sodium 3-nitrobenzenes sulfonate Reference Standard and Test Item Solutions
Sample Weight (mg) Final Volume (mL) Identification Volume made using
Reference Standard 25.47 25 A Milli Q Water Test Item 24.20 25 R 1 

### The preparation of refe rence standard solution A (Refer Section 2.8.1 for Reference Standard) and R 1 

### (Refer Section 2.8.4 for Test Item)

### JRF Study Number: 240-2-11-5551 (Final Report) Page 16 of 52

2.9.2 LC Mass Parameters The Above prepared solutions were anal yzed on LC-MS using following parameters: LC-MS Parameters Instrument : LC-MSD SL Column : C-18, [150 mm x 4.6 mm (i.d.), 3.5 μm particle size] Wave Length : 260 nm Mobile Phase : Acetonitrile (70): 0.1 % Formic Acid (30) Flow Rate : 0.7 mL/minute Injection Volume : 20.0 μL MASS PARAMETERS Mass Range : 50 to 700 m/z Dry Gas Flow Rate : 10.0 L/minute Dry Gas Temperature : 350 °C Nebulizer Pressure : 50 psi Fragmentor Voltage : 100 - 125 Volts (negative) Quadra pole Temperature : 100 °C Capillary Voltage : 4000 Volts (positive and negative) 2.10 Procedure for NMR Analysis
2.10.1 Preparation of Sodium 3-nitrobenzenes sulfonate Reference Standard and Test Item Solution A quantity of 5 mg (approx.) sodium 3-nitroben zene sulfonate reference standard and test item

were transferred separately into the NMR probe tu be and mixed with 1.0 mL deutorated methanol for NMR analysis using following parameter. NMR Parameters Instrument : Avance make Bruker (300 MHz) Atom : H 1 

### Range : Delta 0 – 10 Solvent : Deuterated Methanol (MeOD) Test Site NMR Spectrometer equipment at the Evotec India Li mited, Thane, Mumbai, India. The test site selected for this study is not a member of National GLP Compliance Monitoring Program. The test site was selected in consultation with th e study sponsor. Jai Research Foundation does not claim the GLP compliance for the test site and the part of study conducted.

### JRF Study Number: 240-2-11-5551 (Final Report) Page 17 of 52


### 3. RESULTS 3.1 Infrared Spectra The IR spectral analysis sodium 3-nitrobenzene su lfonate reference standard and test item were confirmed their structure based on the presen ce of specific absorption bands due to the characteristic functional groups: Functional groups Reference Standard Sample - CH stretching 3106.76, 2936.09 and 2873.42 3102.9, 2935.13 and 2873.42 - CH bending 1467.56 1468.53 -C=C stretching in benzene ring 1611.23 1610.27 -NO 2 stretching 1536.99 and 1351.86 1536.02 and 1351.86 -SO 3 H stretching 1190.83, 1051.98 and 647.965 1192.76, 1047.16 and 670.142 -adjacent H- atom 764.637 764.637 

### 3.2 Mass Analysis Characterization of sodium 3-nitrobenzene sulf onate was studied using LC-MS by injecting solutions of sodium 3-nitrobenzene sulfonate reference standard and test item. The characterization data based on molecular mass and retention time are summarized below: Sample Retention Time (min) Mass (m/z) Reference Standard 3.999 202.2 Test Item 4.018 202.2 3.3 NMR Analysis Identification of sodium 3-nitrobenzene sulfonate in reference standard and test item using NMR based on chemical shift and numbers of proton present in standard and test item are summarized below: Molecular Structure:

SO 3

NO 2 H

H H

H Na + -

1

2 3 4

Identification No of Protons Multiplicity Chemical Shift ( δ ), ppm Integration Standard Sample Standard Sample 1 1 Triplet 7.724 – 7.764 7.723 – 7.764 1.09 1.09 2 1 Doublet 8.216 –8.242 8.216 –8.242 1.06 1.06 3 1 Doublet 8.327- 8.369 8.327- 8.369 1.11 1.18 4 1 Singlet 8.666 – 8.675 8.665 – 8.675 1.00 1.00 

### 3.4 Active Ingredient Content Active Ingredient Content of sodium 3-nitrobe nzene sulfonate was carried out using validate analytical method [Reference JRF Research Number: 228-2-13-5312]. The mean active Ingredient Content of sodi um 3-nitrobenzene sulfonate was 96.88% w/w.

### JRF Study Number: 240-2-11-5551 (Final Report) Page 18 of 52


### 4. CONCLUSION From the results of the presence of characteris tic absorption bands revealed in IR spectra, molecular mass and the retention time obtained by LC-MS and the numbers of protons present revealed by NMR spectra the structure of sodium 3-nitrobenzene sulfonate was confirmed and it
was concluded that the sodium 3-nitrobenzene sulfona te technical is similar to that of sodium 3-
nitrobenzene sulfonate reference standard.

### JRF Study Number: 240-2-11-5551 (Final Report) Page 19 of 52


### 5. REFERENCES European Commission: Directorate General H ealth and Consumer Protection 2000: SANCO/ 3030/99 rev. 4 “Technical and Material Prepara tions: Guidance for generating and reporting methods of analysis in support of pre-and post-re gistration data requirements for Annex II (Part A Section 4) and Annex III (part A, Section 5) of Directive 91/414/EEC” as amended by European
Commission Directive 94/37/EC and 96/46/EC.

JRF/PC/SOP-4090: Standard Operating Procedur e for Spectra Analysis (IR, UV, Mass and NMR) of Test Item.

Shrimali Asfak, 2012: “Validation of Analy tical Method for Determination of Sodium 3- Nitrobenzenesulfonate Active Ingredient Concen tration In Dose Formulation”, JRF Research Number 228-2-13-5312, Jai Research Foundatio n, Valvada - 396 108, Gujarat,India.

OECD, 1998: OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1, "OECD Principles on Good Laboratory Practice" ENV/MC/CHEM(98)17 (as revised in 1997).

Robert M. Silverstein, Francis X. Webster, 1997: Spectrometric Identification of Organic Compounds, Wiley, 6 th 

### edition.

U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). United States Environmental Protection Agency, Product Properties Test Guidelines (August 1996).

Williams Dudley H.; and Fleming Ian, Spectroscopic Methods in Organic Chemistry: Tata McGraw-Hill Publishing Company Limited, 4 th 

### Edition (1988).

### JRF Study Number: 240-2-11-5551 (Final Report) Page 20 of 52


### Characterization of Sodium 3-Nitrobenzene Sulfonate (CAS # 127-68-4, US EPA HPV 3)

TABLE 1 Linearity Data of Sodium 3-Nitrobenzene Sulfonate Reference Standard

Concentration (mg/L) Replications Peak Area Counts Me an Peak Area Counts % Variation

24.96 R1 977603 979493.00 0.39 R2 981383

49.92 R1 1964680 1965925.50 0.13 R2 1967171

74.88 R1 3036694 3033790.00 0.19 R2 3030886

99.84 R1 3994671 3991843.50 0.14 R2 3989016

124.80 R1 4969853 4971502.50 0.07 R2 4973152

149.76 R1 5920712 5921661.50 0.03 R2 5922611 Typical Calculation Maximum Peak Area

− Minimum Peak Area % Variation = --------------------------------------------------------- × 100 Maximum Peak Area 981383-977603 = ----------------------- × 100 981383 = 0.39%



### FIGURE 1: Linearity Curve of Sodium 3- Nitrobenzene Sulfonate Reference Standard

0 25 50 75 100 125 150 Concentration (mg/L) 0 900 1800 2700 3600 4500 5400Mean Peak Area (Thousands)

Intercept with y-axis (a) = 8806.63 Slope of the line (b) = 39704.24 Correlation co-efficient or 'r' value = 0.999 Equation : Y = bX + a Y = 39704.24X + 8806.63

### JRF Study Number: 240-2-11-5551 (Final Report) Page 21 of 52


### Characterization of Sodium 3-Nitrobenzene Sulfonate (CAS # 127-68-4, US EPA HPV 3)

TABLE 2 Calculation of Active Ingredient Content

Replication Weight (mg) of Sample

(W) N° ofInjection Peak Area of Sample

(R) Weight (mg) of Standard

(W 1 ) Peak Area of Standard Mean Peak Area of Standard (R 1 ) A.I. Content (% w/w) Mean A.I. Content (% w/w)

R 1 24.20 I 3747663

25.47 3993545

3994632.75 96.77 96.91 II 3758360 3997020 97.04

R 2 24.34 I 3762143 3990825 96.58 96.85 II 3783260 3997141 97.12

Mean 96.88 Purity of Standard (P) 98.00% Dilution Factor (D) 1.00 Typical Calculation Sodium 3-Nitrobenzene Sulfonate A.I. Content (% w/w)

R × W 1 × P = ------------------- × D R 1 × W 3747663 × 25.47 × 98.00 = --------------------------------- × 1.00 3994632.75 × 24.20
= 96.77% w/w

### JRF Study Number: 240-2-11-5551 (Final Report) Page 22 of 52


### Characterization of Sodium 3-Nitrobenzene Sulfonate (CAS # 127-68-4, US EPA HPV 3)

APPENDIX 1 IR, Mass, NMR Spectra and HPLC Chromatograms

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### Characterization of Sodium 3-Nitrobenzene Sulfonate (CAS # 127-68-4, US EPA HPV 3)

APPENDIX 2 Signed Study Plan and Study Plan Amendment

JRF Study Number: 240-2-11-5551 Page 1 of 13

STUDY PLAN
CHARACTERIZATION OF SODIUM 3-NITROBENZENE SULFONATE (CAS # 127-68-4, US EPA HPV 3)
GUIDELINES: SANCO/ 3030/99 rev. 4 AND U.S. EPA OPPTS 830.1800 TEST RULE: 40 CFR 799.5089

SPONSOR

TIARCO CHEMICAL 1300 TIARCO DRIVE DALTON GA 30720

TECHNICAL CONTACT: JEFF VANDERLIP
REGULATORY CONTACT: BENNETT KING
STUDY DIRECTOR: NIRAL M. BHANDARI

TEST FACILITY
JAI RESEARCH FOUNDATION DEPARTMENT OF CHEMISTRY VALVADA - 396 108 DIST. VALSAD GUJARAT

INDIA

OCTOBER - 2012

### JRF Study Number: 240-2-11-5551 (Final Report) Page 36 of 52 APPENDIX 2 (Continued) JRF Study Number: 240-2-11-5551 Page 2 of 13
CONTENTS PAGE N° TITLE PAGE .................................................................................................................... ....................................... 1 CONTENTS....................................................................................................................... ...................................... 2 1. GENERAL INFORMATION ........................................................................................................ ................... 3 1.1 Study Professional ....................................................................................................... .......................... 3 1.2 Test Facility Management ................................................................................................. ..................... 3 1.3 Technical Cont act ........................................................................................................ .......................... 3 1.4 Regulator y Cont act ....................................................................................................... ......................... 3 1.5 Study Schedule ........................................................................................................... ............................ 4 1.6 Study Plan and Amendm ent (if any) Distribu tion ........................................................................... ....... 4 2. INTRODUCTION ............................................................................................................... ............................. 4 2.1 Objective ................................................................................................................ ................................ 4 2.2 Regulatory Guidelines .................................................................................................... ........................ 4 2.3 Test Item ................................................................................................................ ................................ 5 2.4 Referen ce Standard ....................................................................................................... ......................... 6 2.5 Reference Stan dard Retention ............................................................................................. ................... 6 3. GOOD LABORATORY PRACTICE (GLP) ............................................................................................. ...... 6 3.1 GLP Co mpliance ........................................................................................................... ......................... 6 3.2 Standard Operating Procedures (SOPs) ..................................................................................... ............ 7 3.3 Amendment to Study Plan .................................................................................................. ................... 7 3.4 Deviation from the Study Plan ............................................................................................ ................... 7 3.5 Quality Assurance ........................................................................................................ .......................... 7 4. EXPERIMENTAL PROCEDURE ..................................................................................................... .............. 7 4.1 Equipment/Instruments .................................................................................................... ...................... 7 4.2 Solvents and Chemicals ................................................................................................... ...................... 7 4.3 Sample Prepara tions for IR Test .......................................................................................... .................. 7 4.3.1 Interpretation of IR Spectra .......................................................................................... ........... 8 4.4 Methodology for Mass Analysis [b y LC-MS] ................................................................................. ...... 8 4.4.1 Preparation of Reference Standard / Test Item Soluti on for Mass Spectra .............................. 8 4.4.2 Mass Parameters ....................................................................................................... ............... 8 4.4.3 Mass Interpre tation and Reportin g ..................................................................................... ...... 9 4.5 Methodology fo r NMR Analysis ............................................................................................. .............. 9 4.6 Active Ingred ient Content ................................................................................................ ...................... 9 4.6.1 Preparation of Refere nce Standard St ock Solution .................................................................. 9 4.6.2 Li nearity ............................................................................................................. ...................... 9 4.6.3 Preparation of Sample Solution ........................................................................................ ..... 10 4.6.4 Instrumental Parameters ............................................................................................... ......... 10 4.6.5 Calculation of Active In gredient Content (% w/w) ............................................................... 10 5. REPORT ..................................................................................................................... .................................... 11 6. ARCHIVES ................................................................................................................... ................................. 11 7. REFERENCES ................................................................................................................. .............................. 11 8. STUDY PLAN APPROVAL ........................................................................................................ .................. 13

### JRF Study Number: 240-2-11-5551 (Final Report) Page 37 of 52 APPENDIX 2 (Continued) JRF Study Number: 240-2-11-5551 Page 3 of 13

1. GENERAL INFORMATION 1.1 Study Professional

Note: List of additional study personnel will be documented in th e study file and will be presented in the final report.

1.2 Test Facility Management : Hemant Desai Assistant Director, Department of Chemistry Jai Research Foundation, Off. N.H. N° 8 Near Daman Ganga Bridge Valvada. 396 108, Dist. Valsad, Gujarat, India. Ph.: + 91-260 654 0242 (Office) Ph.: +91 99099 00940 (Mobile) E-mail: desaihs@jrfonline.com

1.3 Technical Contact : Jeff Vanderlip 706/277-1300 Tiarco Chemical 1300 Tiarco Drive Dalton. Georgia 30720

1.4 Regulatory Contact : Bennett King 864/457-2321 Tiarco Chemical 1300 Tiarco Drive Dalton. Georgia 30720 Name of Personnel Qualification Profession Experience Training Niral M. Bhandari (Study Director) M.Sc. Chemist 6 years Declar ed Study Director for all Physico-chemical studies

A.H. Patel (Dy. Study Director) M.Sc. Chemist 14 years Declar ed Study Director for all Physico-chemical studies

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1.5 Study Schedule Study Initiation Date : October 12, 2012 Experiment Start Date : October 15, 2012 Experiment Completion : Latest by October, 2012 Draft Report Submission : Latest by October, 2012 Study Completion : Within tw o weeks from the date of r eceipt of the comments on the final draft report from the Sponsor.

1.6 Study Plan and Amendment (if any) Distribution a. Original copy in Archive and Study Sponsor; b. Photocopy to Study Director and QAU

2. INTRODUCTION 2.1 Objective The objective of this study is to determine IR, Mass, NMR and active ingredient content of sodium 3- nitrobenzene sulfonate (CAS N° 127-68-4). 2.2 Regulatory Guidelines European Commission: Directorate General Health and Consumer Protection 2000: SANCO/ 3030/99 rev. 4 “Technical and Material Prepara tions: Guidance for generating and reporting methods of analysis in support of pre- and post-registration data requirements for Annex II (Part A Section 4) and Annex III (part A, Section 5) of Directive 91/414/EEC” as amended by European Commission Directive 94/37/EC and 96/46/EC. U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). United States Environmental Protection Agency, Product Properties Test Guidelines (August 1996).

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2.3 Test Item The Test Item Data Sheet has been completed by the Sponsor. The representative sample of the test item will be retained for archive purpose. Any residual test item will be disposed of at JRF after the expiry date unless otherwise instructed by the Sponsor. The details relating to the test item (as furnished by the study Sponsor) are as follows:

Test Item Name IUPAC Name C.A. Name CAS N° Batch N° Analysed Purity Manufacture by Supplied by Date of Manufacture Date of Expiry Appearance Sodium 3-nitrobenzene sulfonate Benzenesulfonic acid, 3-nitro, sodium salt (1:1) Benzenesulfonic acid, 3-nitro, sodium salt (1:1) 127-68-4 50623 96.9% (Information provided by sponsor via COA) Tiarco Chemical Tiarco Chemical September 08, 2011 September 08, 2013 Yellow to tan granular Data related to the characterization of the test item are documented in a Certif icate of analysis provided by the Sponsor and will be found in annexes of the study report. The test item information and analytical identification provided by the Sponsor will be responsibility of the Sponsor. Storage Condition (at JRF) As per the instruction received from the Sponsor on storage of the test item, the test item will be stored in its original container, as supplied by the Sponsor at 40 - 90 °F in Test Item Control Office (TICO). The stability of the test item in storage will be the responsibility of the Sponsor.

JRF Test Item Code SNB 444

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### SO 3 

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2.4 Reference Standard
Details of the reference standard are as follows:

Reference Standard IUPAC Name C.A. Name Molecular Weight Molecular Formula CAS N° Lot N° Date of Receipt Date of Expiry Purity Source JRF Entry Number Storage Condition Sodium 3-Nitrobenzenes sulfonate Benzenesulfonic acid, 3-nitro-, sodium salt (1:1) Benzenesulfonic acid, 3-nitro-, sodium salt (1:1) 225.15 O 2 NC 6 H 4 SO 3 Na [127-68-4] 07315KNV August 07, 2012 August 06, 2017 (as per JRF SOP-152) 98.0% Sigma-Aldrich, USA CPC-54 Room Temperature
Source: Molecular structure was obtained from Internet.

2.5 Reference Standard Retention The reference standard will be retained at the Department of Chemistry till the declared expiry or re assay date. 3. GOOD LABORATORY PRACTICE (GLP)
3.1 GLP Compliance This study will be conducted in compliance with th e OECD Principles of Go od Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17, N° 1, Environment Directorate, the Organisation for Economic Co-operation and Development, Paris (1998) and all subsequent OECD consensus documents. The NMR spectra analysis will be performed at the Evotec India Limited, Th ane, Mumbai, India (Test Site). The test site selected for this study is not a member of National GLP Compliance Monitoring Program. The test site is selected in consultation with the Study Sponsor. Jai Re search Foundation does not claim the GLP compliance for the test site and the part of study to be conducted.

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3.2 Standard Operating Procedures (SOPs) Unless otherwise specified all procedures mentioned in the study plan are subject to detailed Standard Operating Procedures of Jai Research Foundation. 3.3 Amendment to Study Plan This study plan may be subject to amendment. Amendment to study plan, whether initiated by the Sponsor or the Study Director will be generated, authorized by the Study Director and will be sent to the Sponsor for approval.

In the event that circumstances dictate immediate action, the natu re of these circumstances will communicated to the Sponsor as soon as practicable (by telephone, facsimile transmission or e-mail) and will be confirmed as soon as possible by way of formal study plan amendment. 3.4 Deviation from the Study Plan Any deviation from the study plan will be documented in study file and reported in the study report.
3.5 Quality Assurance This study plan has been verified by JRF QAU and documented (Number 48657). The Quality Assurance Unit (QAU) of JRF will audit/inspect the critical phase(s) of the study, the raw data, draft and final reports. The audit/inspection reports will be provided to the St udy Director and the Test Facility Management. The date of audits/inspections and reporting of findings to the Study Director and the Test Facility Management will be incorporated in the study report.

4. EXPERIMENTAL PROCEDURE
4.1 Equipment/Instruments HPLC, FT-IR Spectrometer, LC-MS, NMR and Balance.

4.2 Solvents and Chemicals Acetonitrile, methanol, Milli-Q water and potassium bromide. 4.3 Sample Preparations for IR Test A known quantity of 2.0 mg reference standard and test item will be mixed with 100 mg (approx.) dried potassium bromide. The mixture will be grinded finely using pestle and mortar and the mixture was placed in DRA sample holder of sample compartment. Th e IR spectrum will be recorded. The background spectrum will be recorded before analyzing the sample by using blank KBr.

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All samples and background spectra will be recorded using the following parameters: Instrument : FT-IR Spectrometer Wave Number Range : 400-4000 cm -1 Resolution : 4.0 cm -1 4.3.1 Interpretation of IR Spectra The spectral data interpretation will be performed using the standard references viz., Williams Dudley H. (1988) and Robert M. Silverstein, Francis X. Webster, (1997).

4.4 Methodology of Mass Analysis [by LC-MS] 4.4.1 Preparation of Reference Standa rd/Test Item Solution for Mass Spectra The known quantities of sodium 3-nitrobenzene sulfonate reference standard and test item will be weighed in into separate volumetric flasks of 10 mL capacity, contents will be dissolved in 5.0 mL Milli-Q water and the volume will be made upto the mark with Milli- Q water. The sample solutions will be injected onto LC-MS in duplicate.

4.4.2 Mass Parameters The Above prepared solution were analysed on LC-MSD SL using following parameters:
LC-MS Parameters

Instrument : LC-MSD SL Column : C-18, [150 mm x 4.6 mm (i.d.), 3.5 μm particle size] Wave Length : 260 nm Mobile Phase : Acetonitrile (70): 0. 1 % formic acid in water (30) v/v Flow Rate : 0.7 mL/minute Injection Volume : 10.0 μL
MASS PARAMETERS Mass Range : 50 to 700 m/z Dry Gas Flow Rate : 10.0 L/minute Dry Gas Temperature : 350 °C Nebulizer Pressure : 50 psi Fragmentor Voltage : 100 - 125 Volts (negative) Quadra pole Temperature : 100 °C Capillary Voltage : 4000 Volts (positive and negative)

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4.4.3 Mass Interpretation and Reporting The mass interpretation will be based on comparison of retention time, elution pattern and mass/fragmentation pattern of standard and test item and the retention time and molecular mass will be reported
4.5 Methodology for NMR Analysis A quantity of 5 mg (approx.) reference standard and test item will be weighed and transferred into the NMR probe tube and mixed with approx. 1.0 mL in suitable solvent for NMR analysis using following parameters: NMR Parameters Instrument : Avance ma ke Bruker (300 MHz) Atom : H 1 Range : Delta 0 – 10 Solvent : Deuterated Oxide (D 2 O) Test Site NMR Spectrometer equipment at the Evot ec India Limited, Thane, Mumbai, I ndia. The test site selected for this study is not a member of National GLP Compliance Monitoring Program. The test site is selected in consultation with the Study Sponsor. Jai Research Foundation does not claim the GLP compliance for the test site and the part of study to be conducted. 4.6 Active Ingredient Content 4.6.1 Preparation of Reference Standard Stock Solution A known quantity of sodium 3-nitrobenzene sulfonate reference standard of known purity will be weighed into a volumetric flask of known capacity. The contents will be dissolved in a small volume of Milli-Q water and the volume will be made upto the mark with Milli-Q water. The various concentrations of reference standard solution will be prepared by dilu tion of stock solution in Milli-Q water for linearity.

4.6.2 Linearity Linearity of the method will be carried out before anal ysing the samples. Six diffe rent concentrations of sodium 3-nitrobenzene sulfonate reference standard w ill be injected onto HPLC in duplicate. The linear calibration curve will be established by plotting the m ean peak areas against concentrations (mg/L). The correlation coefficient (r), intercept (a) and slope (b) will be calculated.

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4.6.3 Preparation of Sample Solution A known quantity of test item will be weighed into a volumetric flask of known capacity. The contents will be dissolved in a small volume of Milli-Q water and the volume will be made upto the mark with Milli-Q water. The solutions will be filtered (if required ) and diluted (if required) using Milli-Q water.

4.6.4 Instrumental Parameters The reference standard solutions, sample solutions and Milli-Q water (solvent used for standard and sample solution preparation) will be injected onto a High Performance Liquid Chromatograph (HPLC) using validated instrument parameters instrumental parame ters [Reference JRF Research Number: RES-2-13- 5312]. Instrument : HPLC Column : C-18 [250 mm x 4.6 mm (i.d.) x 5 μm particle size] Wave length : 260 nm Flow rate : 1.0 mL/minute Injection volume : 20 μL Mobile Phase : Acetonitrile (70): 0.1 % formic acid in water (30) v/v 4.6.5 Calculation of Active Ingredient Content (% w/w) The sodium 3-nitrobenzene sulfonate content will be calculated using the following formula:

R × W 1 × P = -------------- × D R 1 × W Where, R = Peak area of the sample R 1 = Mean peak area of the reference standard W = Weight (mg) of the sample W 1 = Weight (mg) of the reference standard P = Purity (%) of reference standard D = Dilution factor

Note: Active Ingredient content will be conducted in triplicate.

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5. REPORT Unless otherwise requested by the Sponsor, one copy of the final report will be issued along with one soft copy in PDF. The report will include the following information: - Description of the test item and reference st andard including certificates of analysis - Complete details of the experimental procedure - IR spectra with peak tables - Mass Spectra. - NMR Spectra - Spectral interpretation - Summary of observations - Results - Chromatograms - Personnel involved in the study - The signed study plan and study plan amendment(s) (if any) - Record of deviation(s) from the study plan (if any)

6. ARCHIVES On completion of the study all original raw data including any storage medium for electronically recorded data, spectra, documentation, the signed st udy plan, study plan amendment (if any), the draft report, a copy of final report and the representative sample of the test item will be retained in the GLP Archives at Jai Research Foundation for a period of ten years. At the end of this period, the Sponsor's instructions will be sought to either extend the archiving period or return the archived material to the Sponsor or for the material to be disposed of. 7. REFERENCES European Commission: Directorate General Health and Consumer Protection 2000: SANCO/ 3030/99 rev. 4 “Technical and Material Preparations: Guidan ce for generating and reporting methods of analysis in support of pre-and post-registra tion data requirements for Annex II (Part A Section 4) and Annex III (part A, Section 5) of Directive 91/414/EEC” as amended by European Commission Directive 94/37/EC and 96/46/EC. Shrimali A., 2012: “Validation of Analytical Method for Determination of Sodium 3- Nitrobenzenesulfonate Active Ingredient Concentration In Dose Formulation”, JRF Research Number 228-2-13-5312, Jai Research Foundation, Valvada - 396 108, Gujarat, India.

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OECD, 1998: OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1, "OECD Principles on Good Laboratory Practice" ENV/MC/CHEM(98)17 (as revised in 1997). Robert M. Silverstein, Francis X. Webster, 1997 : Spectrometric Identification of Organic Compounds, Wiley, 6 th edition. U.S. EPA, 1996: OPPTS 830.1800 “Enforcement of Analytical Method” (EPA 712-C-96-013). United States Environmental Protection Agency, Product Properties Test Guidelines (August 1996). Williams Dudley H.; and Fleming Ian, Spectroscopic Methods in Organic Chemis try: Tata McGraw-Hill Publishing Company Limited, 4 th Edition (1988).

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### JRF S tudy Numb e

Certifi c r: 240-2-11

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### Characterization of Sodium 3-Nitrobenzene Sulfonate (CAS # 127-68-4, US EPA HPV 3)

APPENDIX 4 Certificate of Analysis of Sodium 3-Nitrobenzenes Sulfonate Standard

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