### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 2 of 43



### STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on the basis of its falling

within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C).

Sponsor T iarco Chemical

1300 T iacro D rive Dalton, GA 30720

Signature

__________________________________

Date

__________________________________

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 6 of 43



### 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

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

SUMMARY

................................................................................................................................................ 10

1. INTRODUCTION

................................................................................................................................ 11 1.1 Study Objective

.......................................................................................................................... 11 1.2 Study Guidelines

........................................................................................................................ 11 1.3 Test Facility and Study Period

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

1.4 Archives

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

2. EXPERIMENTAL PROCEDURE

...................................................................................................... 12

2.1 Test Item

..................................................................................................................................... 12

2.2 Reference Standard

..................................................................................................................... 12 2.3 Instruments/Equipment

.............................................................................................................. 13

2. 4 Solvents and Chemicals

.............................................................................................................. 13 2.5 Principle

...................................................................................................................................... 13

2. 6 Validation of Analytical Method for Determination of Sodium 3-Nitrobenzensulfonate using HPLC

............................................................................................................................... 13

2. 6 .1 Specificity

.......................................................................................................................... 13

2. 6.1.1 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard

Stock Solution

............................................................................................... 13

2. 6.1.2 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard Working Solutions

........................................................................................ 13

2. 6.1.3 Preparation of Test Item Stock Solution

....................................................... 14

2. 6.1.4 Preparation of Test Item Working Solution in Dose Formulation

................ 14

2. 6.2 Instrumental Parameters

................................................................................................ 14

2. 6.3 Limit of Detection (LOD)

............................................................................................ 14

2. 6.3 .1 Preparation of Test Item Solutions in Dose Formulation

............................... 14

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 7 of 43



### CONTENTS (Continued)

PAGE N°

2. 6.4 Linear Dynamic Range

................................................................................................ . 15

2. 6.4 .1 Preparation of Sodium 3-Nitrobenzensulfonate Working Standard Solutions 15

2. 6.5 Precision (% RSD) and Accuracy (% Recovery)

......................................................... 15

2. 6.5 .1 Determination of Homogeneity of Mixing and Stability Sample in Dose

Formulation

................................................................................................... 15

2. 6.6 C alculation

................................ ................................ ................................ .................... 1 6

3. RESULTS

................................ ................................ ................................ ................................ ............ 17

3. 1 Specificity

................................ ................................ ................................ ................................ ... 17

3.2 Linear Dynamic Range

................................ ................................ ................................ .............. 17

3. 3 Limit of Quantitati on

................................ ................................ ................................ ................. 17

3. 4 Precision (% R SD)

................................ ................................ ................................ .................... 17

3. 5 Accuracy (% Recover y)

................................ ................................ ................................ ............ 18

3

. 6 Homogeneity and Stability

................................ ................................ ................................ ........ 18

4. CONCLUSION

................................ ................................ ................................ ................................ .... 19

5. REFERENCES

................................ ................................ ................................ ................................ ..... 2 0

TABLES

1. Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and
Accuracy (% Recovery) and the Level of Test Concentration during
Toxicology Study (0 hr )

............................................................................................................ 21

2. Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of Test Concentration during Toxicology Study ( 24 h )

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

3

. Limit of Quantitation of Sodium 3

- Nitrobenzensulfonate in Dose Formulation

....................... 2 3

4

. Calculation of Precision (% RSD) and Accuracy (% Recovery) of the Level of Test Concentration during Toxicology St udy for Sodium 3

- Nitrobenzensulfonate in Dose Formulation (0 hr)



### ............. 2 4

5



### . Calculation of (%) Recovery and Stability at the Level of Test Concentration during Toxicology Study for Sodium 3

- Nitrobenzensulfonate in Dose Formulation (24h)

.................. 2 5

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 8 of 43

CONTENTS (Continued)
PAGE N°

FIGURES 1. Linear Dynamic Curve for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and Accuracy
(% Recovery) and the Level of Test Concentration during Toxicology Study (0 hr)

............... 21

2. Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of Test Concentration

durin g Toxicology Study ( 24 h )

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

APPENDICES

1. Chromatograms

.......................................................................................................................... 26

2. Signed Study Plan and Study Plan Amendment

........................................................................ 29
3. Certificate of Analysis of Test Item

.......................................................................................... 40
4. Certificate of Analysis of Reference Standard o f Sodium 3-Nitrobenzensulfonate

.................. 41
5. GLP Endorsement of Compliance

.............................................................................................. 42

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 10 of 43



### SUMMARY

This study was conducted to validate the high performance liquid chromatographic (HPLC) analytical

method for the determination of active ingredient concentration of Sodium 3-Nitrobenzensulfonate in dose

formulation

. The test item was supplied by T iarco Chemical, 1300 Tiacro Drive, Dalton, GA 30720

. The

method followed was as per the guidelines of U.S. EPA OPPTS 860.1340 (August, 1996) and

SANCO/3029/99 r ev.4

. The analytical method for the determination of active ingredient concentration of Sodium 3

-

Nitrobenzensulfonate was validated by analysing the t est i tem and reference standard . The validation covered

the aspects namely: (i) specificity, (ii) linear d ynamic range (LDR), (iii) limit of quantitation (LOQ), ( i v)

precision (% RSD) and accuracy (% Recovery).

The method validation are summarised below:

Parameters Results

Dose Formulation Dose Formulation ( RO Water )
Active ingredient Sodium 3

- Nitrobenzensu lfonate
Specificity (Non

- analyte i nterference) No interference

Linear Dynamic Range (LDR) Concentration Range (mg/L) Intercept (a) Slope of the line (b) Correlation Coefficient (r) 10.0 4 to 1 00. 35 12827.10 40335.43 0.999

Limit of Quantitation (LOQ) [mg/ L] 0.5

Precision (% RSD) Fortification Level

-

5 mg/mL 0.07

% RSD [Horwitz equation] 2.99

100 mg/mL 0.85

% RSD [Horwitz equation] 1.90

Accuracy (Recovery %) 5 mg/mL 101.98

100 mg/mL 99.60 

### Homogeneity a nd Stability (Recovery %) 24 Hours 5 mg /mL 101.87

100 mg/mL 99.28

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 11 of 43



### 1. INTRODUCTION
1.1 Study Objective

This study was performed to validate the HPLC analytical method for determination of Sodium 3-
Nitrobenzensulfonate active ingredient concentration in dose formulation. The study was
conducted in compliance with OECD Principles of GLP (1998).

1.2 Study Guidelines

The present study was conducted according to the following guidelines:

U.S. EPA, 1996: OPPTS 860.1340 “ Residue Analytical Method ” . United States Environmental
Protection Agency, Residue Chemistry Test Guidelines, EPA 712-C- 96 -174 (August 1996),

and
European Commission 2000: Residues: Guidance for generating and reporting methods of analysis
in support of pre-registration data requirements for Annex II (part A, Section 4), and Annex III
(part A, Section 5) of Directive 91/414; SANCO/3029/99 rev. 4 of 11 July 2000."Directorate

General Health and Consumer Protection".

1.3 Test Facility and Study Period

This study was performed at the Department of Chemistry, Jai Research Foundation

, Va lvada

– 396 108, Dist. Valsad, Gujarat, India.

Study Initiation : August 25, 2012
Experiment Start : August 30, 2012
Experiment Completion : August 31, 2012
Study Completion : October 11, 2012

1.4 Archives

All original raw data including any storage medium for electronically recorded data,
documentation, chromatograms, the signed study plan, 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
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.

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 12 of 43



### 2. EXPERIMENTAL PROCEDURE

2.1 Test Item

Details of Test Item Provided by the Sponsor

Test Item Name

IUPAC Name

CA Name

CAS Number

Batch/Lot N°
Concentration / Purity
Supplied by
Manufactured by

Date of Manufacture
Date of Expiry
Appearance / Colour Sodium 3

- Nitrobenzensulfonate
Benzenesulfonic acid, 3

- nitro

- ,sodium salt (1:1)
Benzenesulfonic acid, 3

- nitro

- ,sodium salt (1:1)

127

- 68

- 4

50623

9 6.9 % ( Certificate of Analysis, Refer Appendix

- 3 )

Reservol LP

Tiarco Chemical
August 0 9 , 2011
August 0 9 , 201 3
Yellow to tan granular solid

Storage Condition (at JRF) As per the instruction received from the Sponsor on storage of the test item, the test item w as stored in its original container as supplied by the Sponsor in room temperature ( 40 to 90 ° F ) in the Test Item Control Office (TICO) . The stability of the test item in
storage w as the responsibility of the Sponsor.

2.2 Reference Standard

Details of the Reference Standard

Reference Standard

C.A. Name Molecular Weight

Molecular Formula

CAS N°

LOT N° Date of Receipt

Date of Expiry

Pu rity

Source Storage Condition JRF Entry Number Sodium 3

- Nitrobenzensulfonate Benzenesulfonic acid, 3

- nitro

- ,sodium s a lt (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% ( Certificate of Analysis, Ref er Appendix

- 4 )

ALDRICH Room Temperature

CPC

- 54

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 13 of 43

2.3 Instruments/Equipment

Sr. N° Instrument/Apparatus Model Make / Supplier

1 Analytical Balance GR

- 202 Adair Dutt

2 HPLC 2010 C HT Shimadzu

2.4 Solvents and Chemicals

Sr. N° Name Grade Source

1 Acetonitrile HPLC s.d. fine

- chem ltd.

2 Formic acid HPLC Meark

3 Water Milli

- Q Milli

- Q water system

2.5 Principle

The active ingredient concentration of Sodium 3-Nitrobenzensulfonate is determined by high
performance liquid chromatograph on a reversed phase column (C-18) kromosil using acetonitrile :
0.1% formic acid in water (70: 30), v/v as mobile phase and UV detection at 2 60 nm.

2. 6 Validation of Analytical Method for Determination of Sodium 3-Nitrobenzensulfonate using
HPLC
The analytical method for the determination of active ingredient concentration of Sodium 3-

Nitrobenzensulfonate in dose formulation was validated. The validation covered the aspects

namely: (i) specificity, (ii) linear dynamic range (LDR), (iii) limit of quantitation (LOQ), (iv )

precision (% RSD) and accuracy (% Recovery).

2. 6 .1 Specificity

2.6.1.1 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard Stock Solution
A quantity of 10.24 mg Sodium 3-Nitrobenzensulfonate (9 8.0% purity) was weighed in a volumetric

flask of 10 0 mL capacity, dissolved in 10 mL RO water and the volume was made upto the mark with

RO water. The concentration of Sodium 3-Nitrobenzensulfonate in the stock solution was 100 .35
mg/L (standard stock solution RS )

.
2.6.1.2 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard Working Solutions

A volume of 1.0 mL stock solution RS was transferred into volumetric flask of 10 mL capacity and
the volume was made upto the mark with RO water. The solution concentration of Sodium 3-

Nitrobenzensulfonate was 10 .04 mg/L [working solution ( L6 )]

.

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 14 of 43

2.6.1.3 Preparation of Test Item Stock Solution

A quantity of 10.34 mg Sodium 3-Nitrobenzensulfonate technical (96.9% purity) was weighed in a

volumetric flask of 10 0 mL capacity and the volume was made upto the mark with dose formulation
(RO water). The concentration of Sodium 3-Nitrobenzensulfonate in the stock solution was 100.19
mg/L (Test Item stock solution, TI)

.

2.6.1.4 Preparation of Test Item Working Solution in Dose Formulation

A volume of 1 .0 mL t es t item stock solution TI was transferred into volumetric flask of 10 mL
capacity and the volume was made upto the mark with RO water. The solution concentration of

Sodium 3-Nitrobenzensulfonate was 10.02 mg/L [Test Item working solution (STI )]

.

The blank RO water

, Sodium 3-Nitrobenzensulfonate reference standard working solution ( L6 ) and
test item working solution (S TI ) were injected onto HPLC in accordance with section 2.6.2

2. 6.2 Instrumental Parameters [Reference JRF Research Number : RES-2- 12

- 5315]

Instrument : HPLC
Column : C-18 kromosil [250 mm x 4.6 mm (i.d.) x 5 μm particle size]
Wave length : 2 60 nm

Flow rate : 1.0 mL/minute
Injection volume : 20 μL
Mobile Phase : Acetonitrile (70): 0.1 % formic acid in water (30) v/v
Retention Time : 4.0 min. (Appox.)

2. 6.3 Limit of Quantitation (LOQ)

2.6.3 .1 Preparation of Test Item Solutions in Dose Formulation

Stock Solution Solution

Taken (mL) Fina Volume

(mL) Volume

made up with Concentration

Obtained (μg/mL) Identification

of Standard Solutio n TI (100.19 mg/L) 1.0 10

RO water 10.02 LOQTI

LOQTI (10.02 mg/L) 0.1 10 0.10 LOQTI 1

0.5 10 0.50 LOQTI 2 1.0 10 1.00 LOQTI 3 1.5 10 1.50 LOQTI 4



### The test item solutions (LOQ TI 1 

### , LOQ TI 2 

### , LOQ TI 3 

### and LOQ TI 4 

### ) were injected onto the HPLC in
duplicate. The minimum concentration which could be quantified with signal to noise ratio (S/N)

 10:1 was considered as LOQ . The average signal: noise ratio was calculated the noise obtained in

RO water untreated control dose formulation.

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 15 of 43

2. 6.4 Linear Dynamic Range

2. 6.4 .1 Preparation of Sodium 3-Nitrobenzensulfonate Working Standard Solutions

Standard Solution Solution

Taken (mL) Volume make

upto (mL) Obtained

Concentration (mg/L) Solution Standard

Solution

RS ( 100.35 mg/L) - - 100.35 L 6
8.0 10.0 80.28 L 5
6.0 10.0 60.21 L 4
4.0 10.0 40.14 L 3
2.0 10.0 20.07 L 2
1.0 10.0 10.04 L 1



### Above standard solutions were diluted with RO water of un treated control dose formulation.
The reference standard working solutions L 1 

### , L 2 

### , L 3 

### , L 4 

### , L 5 

### and L 6 

### mg/L were injected onto the
HPLC in duplicate and the mean peak area was plotted against concentration (mg/L). The
correlation coefficient (r), slope (b) and intercept (a) were calculated for 0 and 24 hours.

2.6.5 Precision (% RSD) and Accuracy (% Recovery)

2.6.5.1 Determination of Homogeneity of Mixing and Stability Sample in Dose Formulation

The test media samples for homogeneity and stability was treated and provided by Toxicology
Department are as below:

Treated Samples/Group (mg/mL) Replication Theoretical Co ncentration (mg/ L) Theoretical

Concentration Level based on Purity of Test Item (mg/L)

Control Level (0) Blank R1

0.00 0.00

R2 R3 R4 R5 R6

( 5 mg/mL) L R1

5000 4845

L R2 L R3 L R4 L R5 L R6

( 100 mg/mL) H R1

100000 9 6900 H R2 H R3 H R4 H R5 H R6

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 16 of 43

A volume of 0.1 mL from 5 mg/mL solution (LR1, LR2, LR3, LR4, LR5 and LR6) were
transferred into separate volumetric flasks of 10 mL capacity and the volume of each flask was

made up to the mark with RO water. The dilution factor was 100.

A volume of 0. 05 mL from 100 mg/mL solution (HR1, HR2, HR3, HR4, HR5 and HR6) were
transferred into separate volumetric flasks of 10 0 mL capacity and the volume of each flask was

made up to the mark with RO water

. The dilution factor was 2000.

The blank RO water in six replication and above prepared samples were injected onto HPLC in

accordance with section 2.6.2

2. 6

. 6 Calculation

Y

- a

Sodium 3

- Nitrobenzensulfonate a.i. concentration, (mg/L) =
------ x D b Where,

Y = Peak area of the sample

a = Constant

b = Regression co

- efficient for Y on „X‟

D = Dilution factor

The % RSD was calculated using the following formula:

Standard Deviation Precision (% RSD) =
---------------------- ------------------ x 100 Mean Recovered Concentration

The Accuracy (% Recovery) was calculated using the following formula:

Recovered Concentration % Recovery =
-------------------------------- x 100

Fortified Concentration

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 17 of 43



### 3. RESULTS

3.1 Specificity

The specificity of the method for the determination of Sodium 3-Nitrobenzensulfonate active

ingredient concentration in dose formulation was studied by injecting RO water, standard solution

of Sodium 3-Nitrobenzensulfonate and test item solution. Since there was no interference between

the peaks of active ingredient standard, sample as well as solvent (acetonitrile) and untreated dose

formulation , t he method was considered to be specific for the analyte.

3.2 Linear Dynamic Range

The linearity of the method for precision and accuracy was established by injecting six different

concentrations viz. 10.04, 20.07, 40. 14 , 60. 21 , 80. 28 and 100.35 mg/L (Refer Table 1) of Sodium

3-Nitrobenzensulfonate reference standard solution in duplicate and the mean peak area was

plotted against concentration (mg/L). The correlation coefficient (r) was 0.999 (Refer Figure 1 ).

The linearity of the method for stability at the level of dose formulation concentration treated and

provided by the toxicology study was established by injecting the six different concentrations viz.

10.04, 20.07, 40.14, 60.21, 80.28 and 100.35 mg/L mg/L (Refer Tables 2) of Sodium 3-

Nitrobenzensulfonate reference standard solution in duplicate and the mean peak area was plotted

against concentration (mg/L). The correlation coefficient (r) was 0.999 (Refer Figures 2 ).

3. 3 Limit of Quantitation

The limit of quantitation (LOQ) of Sodium 3-Nitrobenzensulfonate was determined by injecting

different fortified in dose formulation at various concentrations by test item

. The minimum signal

to noise ratio  10:1 was considered for LOQ. The lowest quantifiable concentration (LOQ) of

Sodium 3-Nitrobenzensulfonate in dose formulation with signal to noise ratio of 14.88 was 0.50

m g/ L (Refer Table 3 ).

3. 4 Precision (% RSD)

Precision of the analytical method was determined by analysing dose formulation fortified with test

item at the 5 and 100 mg/mL. The precision (% RSD) of analytical method in dose formulation at 5

mg/mL was 0.07 %. The corresponding precision (% RSD) at 100 mg/mL was 0 .8 5 % (Refer

Table 4 ).

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 18 of 43

3. 5 Accuracy (% Recovery)

Accuracy of the analytical method was determined by analysing dose formulation fortified with test

item at the 5 and 100 mg/mL

. The mean accuracy (% recovery) of Sodium 3-Nitrobenzensulfonate

in dose formulation at 5 mg/mL was 101.98 %. The corresponding mean accuracy (% recovery) at

100 mg/mL was 99.60 % (Refer Table 5 ).

3.6 Homogeneity and Stability

The homogeneity and stability of test item in dose formulation was determined by analysing dose

formulation fortified with test item at the level of dose fo rmulation treated and provided by the

toxicology study at different intervals i.e, 0 h and 24 h

. The homogeneity and stability was

calculated as (%) recovery. The results are summarized in table provided below:

Interval 5 mg/mL ( % Recovery) 100 mg/mL ( % Recovery) Replication 0 h 24 h Replication 0 h 24 h

LR1 101.95 101.88 HR1 99.77 99.94

LR2 102.02 101.69 HR2 100.32 98.07

LR3 101.97 101.77 HR3 100.28 99.98

LR4 102.09 101.88 HR4 98.2 3 98.42

LR5 101.99 101.89 HR5 98.92 99.91

LR6 101.86 102.11 HR6 100.08 99.37

Mean 101.98 101.87 Mean 99.60 99.28

SD 3.6572 6.9368 SD 821.0439 813.9644

% RSD 0.07 0.14 % RSD 0.85 0.85

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 19 of 43



### 4. CONCLUSION

From the results of the validation it is concluded that the method is sensitive and precise for the

analysis of active ingredient content concentration, homogeneity and stability of Sodium 3-

Nitrobenzensulfonate in dose formulation. The results of validation criteria are within the guidelines

(U.S. EPA OPPTS 860.1340 and SANCO/3029/99 rev.4) specified limit.

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 20 of 43



### 5. REFERENCES

European Commission 2000: Residues: Guidance for generating and reporting methods of analysis

in support of pre-registration data requirements for Annex II (part A, Section 4), and Annex III

(part A, Section 5) of Directive 91/414; SANCO/3029/99 rev. 4 of 11 July 2000, ” Directorate

General Health and Consumer Protection ”

.

JRF SOP Number: 184 “ Determination of Active Ingredient Content, Stability and Homogeneity of

Test Item in Test vehicle ”

.

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).

U.S. EPA, 1996: OPPTS 860.1340 “ Residue Analytical Method ” . United States Environmental

Protection Agency, Residue Chemistry Test Guidelines, EPA 712-C- 96 -174 (August 1996).

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 21 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

TABLE 1

Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and Accuracy (% Recovery) and the Level of Test Concentration during Toxicology Study (0 hr)

Concentration (mg/L) Replication Peak Area Count Mean Peak Area Count % Variation

10.04 R1 411334 410876.5 0.22

R2 410419

20.07 R1 821539 821439 .0 0.02

R2 821339

40.14 R1 1652262 1649433.5 0.34

R2 1646605

60.21 R1 2434775 2432567.5 0.18

R2 2430360

80.28 R1 3251485 3250852.5 0.04

R2 3250220

100

. 35 R1 4058912 4059743 .0 0.04

R2 4060574
Typical Calculation

Maximum Area

- Minimum Area % Variation =
------------------------------------------ x 100 Maximum Area 411334

 410419

=
---------------- --- ----- - x 100 411334 = 0.22 %



### FIGURE 1
Linear Dynamic Curve for Sodium 3-Nitrobenzensulfonate for Precision (%RSD) and Accuracy (% Recovery) and the Level of Test Concentration during Toxicology Study (0 hr)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 22 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

TABLE 2

Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of

Test Concentration during Toxicology Study ( 24 h)

Concentration (mg/L) Replication Peak Ar ea Count Mean Peak Area Count % Variation

10.04 R1 410777 410674.5 0.05

R2 410572

20.07 R1 817289 818671 .0 0.34

R2 820053

40.14 R1 1650343 1647216 .0 0.38

R2 1644089

60.21 R1 2433453 2437494 .0 0.33

R2 2441535

80.28 R1 3245044 3243800 .0 0.08

R2 3242556

100. 35 R1 4068055 4068151 .0 0.00

R2 4068247
Typical Calculation

Maximum Area

- Minimum Area % Variation =
------------------------------------------ x 100 Maximum Area 410777

 410572

=
---------------- ---- --- - x 100

410777 = 0.05 %



### FIGURE 2
Linear Dynamic Range Data for Sodium 3-Nitrobenzensulfonate at the Level of

Test Concentration during Toxicology Study ( 24 h)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 23 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

TABLE 3

Limit of Quantitation of Sodium 3-Nitrobenzensulfonate in Dose Formulation

Solution Concentration (mg/L) Replication Peak Area Count Mean Peak

Area Mean Noise Signal to Noise Ratio (S/ N) Remark

0.1 I 3804 3798 .0

1298.00 2.93

-

II 3792

0.5 I 19188 19311.5 14.88 LOQ

II 19435

1 .0 I 39375 39717.5 30.6 0

-

II 40060

1.5 I 59004 59110.5 45.54

-

II 59217

Replication Total Area of Noise in Blank (a) N° of Noise in Blank (b) Average = a/b

I 1456 1 1456.00

II 1140 1 1140.00

Average Noise Area of Blank 1298.00

Typical Calculation

Mean Peak Area Signal to Noise Ratio =
------------------------- Average Noise Area Limit of Quantitation

19311.5

---- -- ------- = 1 4.88 1 298.0

0. 5 mg/L

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 24 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

TABLE 4

Calculation of Precision (% RSD) and Accuracy (% Recovery) of the Level of Test Concentration during Toxicology Study for Sodium 3-Nitrobenzensulfonate in Dose Formulation (0 hr)

Treated Dose Level (mg/ mL ) Theoretical

Concentra

tion of Test Item (mg/ L ) Active

Ingredient Concentra

tion (mg/L) Repli

cation Peak

Area of Sample (Y) Recovered

(mg/ L ) Recovery

(%) Mean Conce

ntration (mg/ L ) Mean

Recovery (%) SD %

RSD

Blank (0.00) - - R1 000 ND

-

- - - - R2 000 ND

- R3 000 ND

- R4 000 ND

- R5 000 ND

- R6 000 ND

-

Lower ( 5 ) 5000 4845 R1 2005161 4939.41 101.95

4940.91 101.98 3.6572 0.07 R2 2006586 4942.95 102.02
R3 2005588 4940.47 101.97
R4 2007838 4946.05 102.09
R5 2005995 4 941.48 101.99

R6 2003430 4935.12 101.86

Higher ( 100 ) 100000 96900 R1 1962617 96678.77 99.77

96513.59 99.60 821.0439 0.85 R2 1973416 97214.23 100.32
R3 1972534 97170.5 100.28
R4 1932521 95186.48 98.23
R5 1945900 9584 9.87 98.92

R6 1968726 96981.68 100.08

Typical Calculation Intercept with y

- axis (a)* 12827.10 Slope of the line (b)* 403 3 5.43 Correlation of coefficient (r) 0.999 Purity of Test Item (%) 96.90
Dilution Factor = 10 0 for (L ) and 2 0 00 for ( H )

Concentration (mg/ L ) % Recovery % RSD

Y

– a

=
------- x D b

2005161

– 12827.10

=
----------------------- x 10 0

40335.43

= 4939.41 mg/ L Quantity Recovered

=
------------------------ x 100 Quantity Fortified

4939.41

=
--------- - x 100 4 845

= 101.9 5 % Standard Deviation

=
------------------------ - x 100 Mean Concentration

3.6572

=
----------- x 100 4940.91

= 0.07 %

Key: * = Refer figure 1 page N° 21

ND = Non-Detectable (< Limit of Quantitation i.e., 0. 5 mg/L)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 25 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

TABLE 5

Calculation of (%) Recovery and Stability at the Level of Test Concentration during Toxicology Study for Sodium 3-Nitrobenzensulfonate in Dose Formulation (2 4h )

Treated Dose Level (mg/ mL ) Theoretical

Concentra

tion of Test Item (mg/ L ) Active

Ingredient Concentra

tion (mg/L) Repli

cation Peak

Area of Sample (Y) Recovered

(mg/ L ) Recovery

(%) Mean

Conce

ntration (mg/ L ) Mea

n Recovery (%) SD %

RSD

Blank (0.00) - - R1 000 ND

-

- - - - R2 000 ND

- R3 000 ND

- R4 000 ND

- R5 000 ND

- R6 000 ND

-

Lower ( 5 ) 5000 4845 R1 2003930 4936.28 101.88

4935.62 101.87 6.9368 0.14 R2 2000199 492 7.05 101.69
R3 2001602 4930.52 101.77

R4 2003847 4936.08 101.88
R5 2003963 4936.36 101.89

R6 2008439 4947.45 102.11

Higher ( 100 ) 100000 96900 R1 1965804 96838.05 99.94

96203.24 99.28 813.9644 0.85 R2 1929368 95034.1 1 98.07
R3 1966690 96881.92 99.98
R4 1936051 95364.98 98.42
R5 1965311 96813.64 99.91

R6 1954668 96286.71 99.37

Key: * = Refer figure 2 page N° 22

ND = Non-Detectable (< Limit of Quantitation i.e.,0.5 mg/L)

Dilution Factor = 100 for (L) and 2000 for (H)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 26 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

APPENDIX 1

Chromatograms

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 27 of 43

APPENDIX 1 (Continued)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 28 of 43

APPENDIX 1 (Continued)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 29 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

APPENDIX 2

Signed Study Plan and Study Plan Amendment

JRF Study Number: 228

- 2

- 12

- 5312 Page 1 of 10

STUDY PLAN

VALIDATION OF ANALYTICAL METHOD FOR DETERMINATION OF SODIUM 3

- NITROBENZENSULFONATE ACTIVE INGREDIENT CONCENTRATION IN DOSE FORMULATION

TEST ITEM: SODIUM 3

- NITROBENZENSULFONATE

GUIDELINES: SANCO/3029/99 rev.4 AND U.S. EPA OPPTS 860.1340

SPONSOR

TIARCO CHEMICAL
1300 TIACRO DRIVE DALTON, GA 30720

STUDY DIRECTOR: ASFAK SHRIMALI

TEST FACILITY

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

AUGUST - 2012

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 30 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228-2- 12 -5312 Pa ge 2 of 10

CONTENTS

PAGE N°

TITLE PAGE

........................................................................................................................................................... 1
CONTENTS

............................................................................................................................................................. 2
1. GENERAL INFORMATION

........................................................................................................................... 3 1.1 Study Director

........................................................................................................................................ 3 1.2 Test Facility Management

...................................................................................................................... 3 1.3 Study Schedule

....................................................................................................................................... 3 1.4 Study Plan and Amendment (if any) Distribution

.................................................................................. 3
2. INTRODUCTION

............................................................................................................................................ 3 2.1 Objective

................................................................................................................................................ 3 2.2 Regulatory Guidelines

............................................................................................................................ 3
2.3 Test Item

................................................................................................................................................ 4
2.4 Reference Standard

................................................................................................................................ 5
3. GOOD LABORATORY PRACTICE (GLP)

................................................................................................... 5 3.1 GLP Compliance

.................................................................................................................................... 5 3.2 Standard Operating Procedures (SOPs)

................................................................................................ . 5 3.3 Amendment to Study Plan

..................................................................................................................... 5 3.4 Deviation from the Study Plan

............................................................................................................... 6 3.5 Quality Assurance

.................................................................................................................................. 6
4. EXPERIMENTAL PROCEDURE

.................................................................................................................. 6 4.1 Equipment/Instruments

......................................................................................................................... 6
4.2 Solvents and Chemicals

........................................................................................................................ 6 4.3 Validation of the Analytical Method for Sodium 3-Nitrobenzensulfonate by HPLC

........................... 6 4.3.1 Specificity

............................................................................................................................... 6 4.3.2 Linear Dynamic Range (LDR)

............................................................................................... 7 4.3.3 Limit of Quantitation (LOQ)

.................................................................................................. 7 4.3.4 Precision (% RSD) and Accuracy (% Recovery) from Test Vehicle

...................................... 7 4.3.4.1 Calculation of % Recovery (Accuracy)

.................................................................... 7 4.3.4.2 Calculation of Precision (% RSD)

............................................................................ 7
4.3.5 Instrumental Parameters

......................................................................................................... 8 4.3.6 Calculation of Active Ingredient Concentration

..................................................................... 8 4.3.7 Sample Analyses for Determination of Homogeneity and Stability

....................................... 8
5. REPORT

.......................................................................................................................................................... 8

6. ARCHIVES

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

7. REFERENCES

................................................................................................................................................ 9
8. STUDY PLAN APPROVAL

......................................................................................................................... 10

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 31 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228

- 2

- 12

- 5312 Page 3 of 10

1. GENERAL INFORMATION

1.1 Study Director : Asfak Shrima li

Deputy Study Director : Ashok S. Amruskar

1.2 Test Facility Management : Hemant Desai

1.3 Study Schedule
Study Initiation Date : August 25, 2012
Experiment Start Date : August 30, 2012
Experiment Completion : Latest by August 2012

Draft R eport Submission : Latest by September 2012
Study Completion : Within two weeks from the date of receipt of comments on the
final draft report from the Sponsor.

1.4 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 validate the analytical method for determination of active ingredient

concentration of Sodium 3-Nitrobenzensulfonate in the dose formulation.

2.2 Regulatory Guidelines

European Commission 2000: Residues: Guidance for generating and reporting methods of analysis in support

of pre-registration data requirements for Annex II (part A, Section 4), and Annex III (part A, Section 5) of

Directive 91/414; SAN CO/3029/99 rev. 4 of 11 July 2000, “Directorate General Health and Consumer

Protection”.

U.S. EPA, 1996: OPPTS 860.1340 “Residue Analytical Method”. United States Environmental Protection

Agency, Residue Chemistry Test Guidelines, EPA 712-C- 96 -174 (August 1996).

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 32 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228-2- 12 -5312 Page 4 of 10



### 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

CA Name

CAS Number

Batch/Lot N°
Concentration / Purity
Supplied by

Manufa ctured by

Date of Manufacture
Date of Expiry
Appearance / Colour Sodium 3

- Nitrobenzensulfonate
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 Co A)

Reservol LP

Tiarco Chemical
August 09, 2011
August 09, 2013
Yellow to tan granular solid

Data related to the characterization of the test item are documented in a Certificate 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 in room temperature (40 to 90 °F) in the 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

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 33 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228-2- 12 -5312 Page 5 of 10

2.4 Reference Standard

Reference Standard

C.A. Name
Molecular Weight

Molecular Formula

CAS N°

LOT N°
Date of Receipt
Date of Expiry
Purity

Source
Storage Condition
JRF Entry Number Sodium 3

- Nitrobenzensulfonate
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%

ALDRICH
Room Temperature

CPC

- 54

3. GOOD LABORATORY PRACTICE (GLP)

3.1 GLP Compliance
This study will be conducted in compliance with the OECD Principles of Good Laboratory Practice (as revised
in 1997), ENV/MC/CHEM (98)17, N° 1, Environment Directorate, the Organisation Economic Co-operation
and Development, Paris (1998) and all subsequent OECD consensus documents.

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 nature 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.

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 34 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228

- 2

- 12

- 5312 Pag e 6 of 10

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 47582). 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 Study Director and the Test Facility Management. The date of

audits/inspections a nd 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
Analytical Balance, HPLC and Refrigerator.

4.2 Solvents and Chemicals
Acetonitrile, formic acid, RO water and Milli-Q Water.

4.3 Validation of the Analytical Method for Sodium 3-Nitrobenzensulfonate by HPLC
The analytical method for the determination of Sodium 3-Nitrobenzensulfonate concentration in dose

formulation will be validated by analysis of test item and blank test vehicle. The validation will cover the

aspects namely (i) specificity, (ii) linear dynamic range (LDR), (iii) limit of quantitation (LOQ) (iv), precision

(%, RSD) and accuracy (%, Recovery) from test vehicle (RO Water ).

4.3.1 Specificity
The test item solution, reference solution and blank test vehicle (RO Water) and injected onto a HPLC using

selected parameters. The interference (if any), in the determination of active ingredient in test item will be

reported. Interference should not exceed 30% peak area of the target active ingredient at LOQ level.

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 35 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228-2- 12 -5312 Page 7 of 10

4.3.2 Linear Dynamic Range (LDR)
Six different concentrations of Sodium 3-Nitrobenzensulfonate will be prepared and injected onto HPLC in two

replicates. The linear calibration curve will be established by plotting the mean peak area against concentration

(mg/L). The correlation coefficient (r), intercept (a) and slope (b) will be calculated. The regression equation

will be established.

4.3.3 Limit of Quantitation (LOQ)
The minimum quantity of test item (Sodium 3-Nitrobenzensulfonate ) fortified in the test vehicle, which could be

produced a signal to noise ratio  10:1, will be considered as limit of quantitation (LOQ).

4.3.4 Precision (% RSD) and Accuracy (% Recovery) from Test Vehicle

Precision and accuracy will be established at the level of test concentration used during the toxicology study.
Six determinations will be made at each fortification level. The sample will be diluted, if necessary and
analysed by the HPLC.

The active ingredient concentration in each replicate, mean of active ingredient concentration, SD and % RSD

will be calculated and reported. Each replicate of sample solution will be injected in duplicate. The test item

will be fortified in test vehicle will be injected onto HPLC in accordance with the parameters described in

section 4.3.5

.

4.3.4.1 Calculation of % Recovery (Accuracy)

Recovered Concentration % Recovery (Accuracy) = -------------------------------- x 100 Fortified Concentration

Accuracy/recovery values should fall within 70 to 110%. If the majority of recoveries do not fall within the

expected range the reason will be identified and corrections will be made to the method and if required, th e
validation will be repeated.

4.3.4.2 Calculation of Precision (% RSD)

Precision will be defined by the relative standard deviation (% RSD). The % RSD will be calculated at each

fortification level. The %RSD should be  20% at each level of fortification. The precision (% RSD) will be

calculated as follows:

Standard deviation Precision (% RSD) = --------------------------------------------- x 100 Mean Recovered Concentration

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 36 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228

- 2

- 12

- 5312 Page 8 of 10

4.3.5 I nstrumental Parameters [Reference JRF Research Number: RES

- 2

- 12

- 5315]

Instrument : HPLC

Column : C

- 18, [250 mm x 4.6 mm (i.d.), 5 μm particle size]

Mobile Phase : Acetonitrile : 0.1% Formic acid in Water (70 : 30) v/v
Wave Length : 260 nm

Flow Rate : 1.0 mL/minute
Injection Volume : 20 μL

4.3.6 Calculation of Active Ingredient Concentration

Y = bX + a

Y

- a A.I. Concentration (X) =

-------- x D b Where,

Y = Peak area of the sample

a = Constant

b = Regression Coe fficient for Y on X

D = Dilution factor (if applicable)

4.3.7 Sample Analyses for Determination of Homogeneity and Stability

The dose formulation samples for homogeneity and stability will be prepared and provided by Toxicology
Department at two concentra tion levels along with control according to the procedure described in standard
operating procedure JRF/TOX/288. All the samples will be stored at room temperature during analyses. For the
stability, the samples will be analysed at the interval of 0 and 24 hrs.

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 standard

- Complete deta ils of the experimental procedure

- 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)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 37 of 43
APPENDIX 2 (Continued)

JRF Study Number: 228

- 2

- 12

- 5312 Page 9 of 10

6. ARCHIVES

On completion of the study, all original raw data including any storage medium for electronically recorded data,

documentation, chromatograms, the signed study 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 2000: Residues: Guidance for generating and reporting methods of analysis in support

of pre-registration data requirements for Annex II (part A, Section 4), and Annex III (part A, Section 5) of

Directive 91/414; SANCO/3029/99 rev. 4 of 11 July 2000. "Directorate General Health and Consumer

Protection".

JRF SOP Number: 184 “Validation of Method and Determination of Active Ingredient Concentration, Stability

and Homogeneity of Test Item in Dose Formulation/Test Media”.

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).

U.S. EPA, 1996: OPPTS 860.1340 "Residue Analytical Method". United States Environmental Protection

Agency, Residue Chemistry Test Guidelines, EPA 712-C- 96 -174 (August 1996).

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 38 of 43
APPENDIX 2 (Continued)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 39 of 43
APPENDIX 2 (Continued)

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 40 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

APPENDIX 3

Certificate of Analysis of Test Item

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 41 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

APPENDIX 4

Certificate of Analysis of Reference Standard of Sodium 3-Nitrobenzensulfonate

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 42 of 43



### Validation of Analytical Method for Determination of Active Ingredient Concentration of

Sodium 3-Nitrobenzensulfonate in Dose Formulation

APPENDIX 5

GLP Endorsement Compliance

### JRF Study Number: 228-2- 13 -5312 (Final Report) Page 43 of 43

APPENDIX 5 (Continued)