JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 2 of 85



### 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

__________________________

Signature

__________________________

Sponsor/Submitter

Date

___________________________JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 6 of 85



### 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

CO NTENTS

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

PERSONNEL INVOLVED IN THE STUDY

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

LIST OF ABBREVIATIONS

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

SUMMARY

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

1. INTRODUCTION

.................................................................................................................. 12 1.1 Study Objective

........................................................................................................... 12
1.2 Regulatory Guidelines

................................................................................................ . 12
1.3 Test Method Principle

................................................................................................ . 12
1.4 Test Facility and Study Period

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

1.5 Archives

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

2. EXPERIMENTAL PROCEDURE

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

2.1 Test Item

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

2. 2 Instruments and Equipment

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

2. 3 Solvent and Chemical

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

3. ANIMAL WELFARE

............................................................................................................ 14 3.1 Human Endpoint

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

4. TEST SYSTEM

...................................................................................................................... 15 4.1 Test System Specification

........................................................................................... 15 4.2 Justification of Test System

........................................................................................ 15 4.3 Location of the Study

.................................................................................................. 15 4.4 Experimental Room Sanitation

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

4 .5 Acclimatization

........................................................................................................... 15 4.6 Experimental Conditions

............................................................................................. 15 4.7 Housing

....................................................................................................................... 16

4.8 Diet and Water

............................................................................................................ 16 4.9 Allocation to Groups

................................................................................................... 16

4.10 Animal Identification

.................................................................................................. 16JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 7 of 85

CONTENTS (Continued) PAGE N°

5. EXPERIMENTAL PROCEDURE

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

5.1 Ve hicle and Justification for Choice

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

5.2 Route and Mode of Test Item Administration

............................................................. 17 5.3 Test Formulation Preparation

...................................................................................... 17 5.4 Dosing

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

5. 5 Method of Chemical Analysis

..................................................................................... 17 5.6 Duration of Dosing

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

5.7 Dose Levels and Dose Justification

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

6. OBSERVATIONS

.................................................................................................................. 19 6.1 Clinical Signs

.............................................................................................................. 19 6.2 Mortality and Morbidity

.............................................................................................. 19 6.3 Body Weight

............................................................................................................... 19 6.4 Feed Consumption

....................................................................................................... 19 6.5 Gross Pathological Examination

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

7. DATA AND REPORTING

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

7.1 Tabulation

................................................................................................................... 19 7.2 Statistical Analysis

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

8. RESULTS

............................................................................................................................... 20 8.1 Chemical Analysis (Dose Formulation Analysis)

....................................................... 20

8.2 Mortalities

................................................................................................................... 20

8.3 Clinical Observations

.................................................................................................. 20 8.4 Body Weight

............................................................................................................... 20 8.5 Feed Consumption

....................................................................................................... 20 8.6 Organ Weight

.............................................................................................................. 21 8.7 Macroscopic Pathological Findings

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

9. CONCLUTION

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

10. REFERANCES

....................................................................................................................... 23

TABLES

1. Mortality Record

......................................................................................................... 24

2. Cl inical Signs- Summary By Group

............................................................................ 25

3. Body Weight (g )

– Group Mean Values

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

4. Mean Body Weight Change (%)

– Group Mean Values

............................................. 27

5. Feed Consumption (g/rat/day )

– Group Mean Values

................................................ 28JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 8 of 85

CONTENTS (Continued) PAGE N° FIGURES

1. Mean Body Weight (g) G1 To G4

– Male

.................................................................. 29

2. Mean Body Weight (g) G1 To G4

– Female

............................................................... 30

3. Mean Body Weight Change (%) G1 To G4

– Male

.................................................... 31

4. Mean Body Weight Change (%) G1 To G4

– Female

................................................ 32

5. Mean Feed Consumption (g/rat/day) G1 To G4

– Male

............................................. 33

6. Mean Feed Consumption (g/rat/day) G1 To G4

– Female

.......................................... 34

APPENDICES

1. Record of Animal Fate and Disposition

...................................................................... 35
2. Clinical Signs of Individual Rat

.................................................................................. 37
3. Body Weight (g) of Individual Rat

.............................................................................. 41

4. Mean Body Weight Change (%) of Individual Rat

..................................................... 43

5. F ee d Consumption (g/rat/day) of Individual Rat

........................................................ 45

6. Pathology Report

......................................................................................................... 46

7. Dose Formulation

........................................................................................................ 58
8. Analytical Report on Residues of Organochlorine and Organophosphorus
Pesticides and Heavy Metals in F ee d, Water and Paddy Husk

................................... 62

9

. Results of Microbiological Analysis of Rat Pellet F ee d, Water and Paddy Husk

....... 65

10 . Signed Study Plan

....................................................................................................... 69
11. Certificate of Analysis of Sodium 3-nitrobenzensulfonate

......................................... 83

12

. GLP Endorsement of Compliance

............................................................................... 84JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 10 of 85



### LIST OF ABBREVIATIONS

BMR - Barrier Maintained Rodent

b. wt. - Body weight

F - Female

g - Gram

G - Group

G1 - Control (G ro up given reverse osmosis water alone)

G2 - Low dose (10 mg/kg b. wt./day)

G3 - Mid dose (100 mg/kg b. wt./day)

G4 - High dose ( 10 00 mg/kg b. wt./day)

JRF - Jai Research Foundation

M - Male

mL

- Millilitre

mg

- Milligram

NA

- Not Applicable

n

- Number of Cages

N

- Number of animals/ observations

N°

- Number

NAD

- No abnormalities detected

OECD

- Organisation for Economic Co

- operation and Development

PE /PT

- Pre

- exposure /Pre

- treatment

SD

- Standard D eviation

TOX

- Department of Toxicology

TS

- Terminal S acrifice

T I CO

- Test Item Control Office

T I DS

- Test Item Data Sheet

%

- Percent

°C

- Degree C entigrade

- - Not ApplicableJRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 11 of 85



### SUMMARY

This study was conducted to determine the doses for reproduction/developmental toxicity screening test of sodium 3-nitrobenzensulfonate (sponsored by Tiarco Chemical, Dalton ) in Wistar rats, when administered through oral gavage for a period of 14 consecutive days

. The methods followed were inspired from the G uidelines of the OECD N° 407 (October 3, 200 8)

. Total 20 male and 20 female Wistar rats were randomly divided into four groups. Three dose levels of sodium 3-nitrobenzensulfonate were used at 10 (G2-Low dose), 100 (G3-Mid dose) and 1000 (G 4-High dose) mg /kg b. wt ./day

. A concurrent control group of rats was given reverse osmosis water only. The fix dose volume of 10 mL/kg body weight was used. The dose formulations were prepared by dissolving the
test item in reverse osmosis water

. The dose formulations were analysed for active ingredient (a.i.) concentration and homogeneity at the initiation of treatment (day 1).

All rats were observed once a day for clinical signs and twice a day for mortality and morbidity throughout the study period. Body weight and f ee d consumption of individual animal were determined twice weekly throughout the treatment period

. All the surviving rats were sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination at the end of the treatment period. Absolute organ weights were recorded and relative organ weights were calculated for the organs defined in study plan.

The findings from the present study were:

The analysis result revealed that the mean calculated concentration was within the acceptance level of ±
10% of nominal concentration.

No mortality was observed from any of the groups (G1 to G4) during the study period. All animals belonging to groups G1 to G4 were found normal throughout the study period.

No significant changes were observed in mean body weight

, percent body weight change and f ee d consumption of rats belonging to groups G2, G3 and G4 when compared with the G1 rats.

No external and internal gross lesions were observed in across all the groups. Absolute and relative organ weights were comparable with concurrent control group.

Based on the results of this study, it is concluded that sodium 3-nitrobenzensulfonate did not produce any
adverse effect up to the dose level of 1000 mg/kg b. wt /day when administered through oral gavage for 14 consecutive days in Wistar rats under the conditions and procedures followed in the present study. Based on the result of this dose range finding study, following dose levels are proposed for definitive study:

Dose levels Proposed Dose Level

- I Proposed Dose Level

- I I

Low 10 62.5
Mid 100 250 High 1000 1000JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 12 of 85



### 1. INTRODUCTION

1.1 Study Objective

This study was conducted to determine the doses for Reproduction/Developmental toxicity screening test of sodium 3-nitrobenzensulfonate in Wistar rats.

1.2 Regulatory Guidelines

This study was conducted based on following guidelines with exception of duration of treatment and Clinical Pathology and Pathology:

The United States Environmental Protection Agency (July 200 0). Health Effects Test Guidelines,
OPPTS 870. 3050 , EPA 712-C- 00

- 366, “Repeated Dose 28

- Da y Oral Toxicity Study in Rodents”;

and
Guidelines of Organization for Economic Co-operation and Development (OECD) for Testing of Chemicals, N° 407, entitled “ Repeated Dose 28-Day Oral Toxicity Study in Rodents ” , adopted by the Council on October 03, 2008.

1.3 Test Method Principle

The test item, sodium 3-nitrobenzensulfonate was administered orally by gavage for 14 days to 3 groups of male and female rats. A concurrent control group of male and female rats were given reverse osmosis water only. During the experimental period, all the animals were observed closely for signs of toxicity. At the end of the treatment period, all the surviving animals were humanely sacrificed and subjected to gross examination.

1. 4 Test Facility and Study Period

The study was performed at the Department of Toxicology, Jai Research Foundation, Valvada - 396 108, Dist. Valsad, Gujarat, India.

Study Initiation : October 10, 2012 Experiment Start : October 12, 2012

Terminal Sacrifice : November 01, 2012 Experiment Completion : November 01, 2012 Study Completion : December 26, 2012

1. 5 Archives

All original raw data including any storage medium for electronically recorded data, documentation, the signed study plan, the draft report, a copy of the final report, 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: 410 -1- 04

- 5132 (Final Report) Page 13 of 85



### 2. EXPERIMENTAL PROCEDURE

2.1 Test Item
The details relating to the test item (as furnished by the Study Sponsor) are as follows:

Test Item Name : Sodium 3

- nitrobenzensulfonate

IUPAC Name : Benzenesulfo nic acid, 3

- nitro

- , sodium salt (1:1)

CA Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CAS Number : 127

- 68

- 4 Analysed Concentration : 96.9% ( Refer Certificate of Analysis in Appendix N ° 11)

Batch N° : 50623 Supplied by : Reservol LP

Manufacture d By : Tiarco Chemical

Date of Manufacture : September 08, 2011 Date of Expir y : September 08, 2013 Appearance : Yellow to tan granular solid Storage Conditions (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 room temperature (15

- 30 °C) in the Test Item Control Office (TICO). The stability of the test item in storage will be the responsibility of the Sponsor.

Data related to the c haracterization of the test item were documented in a Certificate of analysis provided by the Sponsor and are included in A ppendix N° 11 of the study report. The test item information and analytical identification provided by the Sponsor were considered as an adequate description of the characterization, purity and stability of the test item and will be responsibility of the Sponsor.

2.2 Instruments and Equipment

Tattoo Machine : Animal Identification & Marking System, Inc. 252 Main Street, Hornell, New York

Balance : Sartorius AG, Germany TE 2101(Capacity

– 0.1 g to 2100 g) Magnetic Stirrer : DBK Instruments Interlink, Mumbai, India

Metal Cannula : IMS Dane Mill, ILA, England ( 16 Gauze x 5 cm) Glass Syringe : Borosilicate Hard Glass Syringe (5 or 3 mL) Safeway Syringe : Safeway syringes (3 mL)JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 14 of 85

2.3 Solvent and Chemical

Disinfectant Solution : Dettol Surface Disinfectant Solution 2.5 %, Reckitt Benckiser (India) Limited

Germ Free Hand Sanitizer (65% Ethanol in Gel Base) : Median Biopharma, Mumbai.

Vehicle : Reverse osmosis water



### 3. ANIMAL WELFARE

Th is study was carried out in compliance with the „Guidelines for Laboratory Animals Facility‟ issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. These guidelines promote the humane care of animals used in research by providing specifications that has been enhancing animals well being and experimental quality for the advancement of biological knowledge that is relevant to humans and animals.

Jai Research Foundation is committed to enhance animal welfare and ensures that studies are designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific obj ectives and in accordance with Jai Research Foundation‟s policy on animal

welfare.

Project proposal for the experimentation subjected to approval by Institutional Animal Ethics Committee (IAEC), Jai Research Foundation.

JRF is also accredited with AAALAC (Association for Assessment and Accreditation of Laboratory Animals Care) that promotes humane care of animals during the experiments.

3 .1 Humane Endpoint

Moribund animals or animals obviously in pain or showing signs of severe and enduring distress shall be humanely sacrificed and are considered in the interpretation of the study results in the same way as animals that died in experiment.JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 15 of 85



### 4. TEST SYSTEM 4.1 Test System Specification

Healthy, young adult rats ( Rattus norvegicus ) of Wistar strain were ob tained from the Animal Breeding Facility, Jai Research Foundation for the use in study. Female rats were nulliparous and non-pregnant. At the initiation of dosing rats were 7 to 8 weeks old.

4 .2 Justification of Test System

Rat is selected as a test system because it is a readily available laboratory rodent species. This species has been historically shown to be a suitable model for repeated dose toxicity studies. The OECD, EPA and other regulatory authorities also recommend it. In addition, a large amount of background knowledge was accumulated on this strain.

4.3 Location of the Study During the study period, the experimental animals were kept in the Barrier Maintained Rodent (BMR N° 7) Facility.

4.4 Experimental Room Sanitation
Each day floor and all worktops were swept and mopped with a disinfectant solution.

4 .5 Acclimatization
A larger number of animals than necessary for the study were ordered to permit the selection and/or replacement of individuals before the start of the treatment. A total of 25 male and 25 female rats were selected for acclimatization. After acclimatization, 20 males and 20 females were selected for the experiment. Remaining 5 male and 5 female were returned to Animal Breeding Facility, Jai Research Foundation. Animals were received into the experimental room and acclimatized for a period of 6 days prior to the start of dosing. During acclimatization period, all animals were observed daily for clinical signs once daily. During acclimatization, animals were marked with temporary identification number on the base of the tail using marker pen.

4.6 Environmental Conditions
Rats were maintained in Barrier Maintained Rodent Facility in an environment controlled room (BMR Room N° 7). The photoperiod was 12 hours fluorescence lighting and 12 hours darkness; light hours were 06.00 - 18.00 hours, approximately. Environmental condition during study period was as given in below table:

Environmental Parameters Temp erature (°C) Relative H umidity (%) Air C hange

(per hour) Mean Light

Intensity (LUX) Max imum Min imum Max imum Min imum

October 2012 23 20 66 64 16 291

The experimental room was cleaned and mopped daily with 2.5% Dettol surface disinfectant.JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 16 of 85

4.7 Housing

Animals were housed in groups of 2 to 3 rats/cage/sex in solid floor polypropylene rat cages (size: 410 mm x 282 mm x 150 mm) during acclimatization and treatment period. Each cage was fitted with a stainless steel top grill having provision for keeping rat pellet f ee d and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks. Cages were changed daily. The cages and water bottles were cleaned in water, dried and then sterilized. After sterilization, they were stored in the UV-room inside the Barrier Maintained Rodent Facility till used. The bottom of the cages was layered with clean sterilised paddy husk as a bedding material. Paddy husk has been analysed for commonly found contaminants at every six months interval and the most recent result is incorporated in the final report (Refer Appendices 8

and 9 ).

4.8 Diet and Water
The experimental animals were fed ad libitum with standard rodent pellet diet (obtained from Teklad Certified Global 16% Protein Rodent diet manufactured by Harlan, USA) with an unlimited supply of clean and filtered drinking water (filtered through Reverse Osmosis water filtration system) in autoclave sterilised polypropylene bottles.

Quality of f

ee d and water (microbial and chemical contaminant analysis) is being checked and monitored at every six months interval in JRF. The most recent analysis reports are incorporated in the final report (Refer Appendices 8 and 9 ).

4.9 Allocation to Groups
Prior to randomization, the animals were weighed. Only healthy rats were allocated to the study. Rats were randomly allocated to different groups using an in-house developed validated computer software program (Gad and Weil, 1994). Animals were divided into four groups G1 (control), G2 (low dose), G3 (mid dose) and G4 (high dose). At the start of the treatment, the body weight variations among the animals were within ± 20 % of the mean body weight for each sex.

4.10 Animal Identification

Individual animal was identified with individual number tattooed at the base of tail after randomization and each cage with a coloured label showing Study N°, Test Item Code, Group N° & Sex, Dose, Study Code, Cage N° and Animal N°.JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 17 of 85



### 5. EXPERIMENTAL PROCEDURE

5.1 Vehicle and Justification for Choice

The reverse osmosis water was selected as a vehicle based on solubility check performed at JRF and after consultation with the sponsor.

5.2 Route and Mode of Test Item Administration

The oral route of administration wa s selected for this study since it is an exaggerated model f or the reasonably foreseeable exposure in human.

5 .3 Test Formulation Preparation

The test item were mixed with RO water to obtain the desired concentration. The test solutions were prepared every day prior to dosing and were used within 4 hours. Prior to dosing, the test solutions were thoroughly mixed using magnetic stirrer and during dosing, the test solutions were mixed intermittently in order to administer a homogeneous preparation.

5.4 Dosing

Test solutions were administered once daily, by gavage, at approximately the same time each day, using intubation cannula attached to a graduated syringe. A constant dose volume of 10 mL/kg were used and individual doses were adjusted according to the most recently recorded body weight of animal. The control group animals were given vehicle only

.

5 .5 Method of Chemical Analysis

The homogeneity and stability of test item in vehicle has been established in JRF Study N° 228-

2- 13

- 5312 following the validated method. The dose formulation were analysed for active ingredient (a.i.) concentration and homogeneity at the initiation of the treatment period of the study. Details are reported in the Appendix N° 7.

5.6 Duration of Dosing

The duration of dosing was 14 days which is selected after consultation with Sponsor. The first day of dose administration was designated as day 1 for each animal.

5.7 Dose Levels and Dose Justification

Based on results of acute oral toxicity study with same test item (JRF Study N° 401-1- 04 -5131), the following three dose levels are selected after consulting with Sponsor.

Dose levels for study was (low dose - 10 , mid dose - 100 and high dose - 1000 mg/kg b. wt./day)

of the test item.JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 18 of 85

Table 1: Grouping and Animal Identification after Randomization

Group N° Group Dose

(mg/kg b. wt./day) Number of Animals Animal

Identification Number Cage Number

M F M F M F

G1 Control 0 5 5 1

- 5 6

- 10 1

- 2 3

- 4

G2 Low Dose 10 5 5 11

- 15 16

- 20 5

- 6 7

- 8

G3 Mid Dose 100 5 5 21

- 25 26

- 30 9

- 10 11

- 12

G4 High Dose 1000 5 5 31

- 35 36

- 40 13

- 14 15

- 16JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 19 of 85



### 6. OBSERVATIONS

Following observations were recorded:

6 .1 Clinical Signs
All animals were observed once a day, preferably at the same time each day.

6 .2 Mortality and Morbidity
All animals were checked twice a day for mortality and signs of morbidity. All rats were sacrificed and subjected to post-mortem examination.

6 .3 Body Weight
Body weight were recorded individually for all animals on the day of randomization, at the beginning of the treatment (i.e., day 1) and twice weekly (i.e., days 1

, 4, 8 and 11 ) and on the day of necropsy (day 15)

.

6 .4 F ee d Consumption
The feed consumption was determined twice weekly and on same intervals as body weight recording.

6 .5 Gross Pathological Examination
All animals were euthanised by carbon dioxide asphyxiation and subjected to a full gross necropsy under the direct supervision of a veterinary pathologist at the end of the treatment period.

All animals were examined carefully for external abnormalities. The thoracic and abdominal cavities were cut opened and a thorough examination of the organs was carried out to detect
abnormalities.

Organ weight were recorded for the testes and epididymis for all male animals.



### 7. DATA AND REPORTING

7 .1 Tabulation
All study observations were recorded in standard formats.

7 .2 Statistical Analysis
Department of Bio-statistics and Systems Information, Jai Research Foundation process ed the raw data to get group means and standard deviations with significance between the control and treated groups, using in-house developed statistical software. The data were summarised in tabular form. All the parameter characterised by continuous data such as body weight, feed consumption, organ weight were subjected to Bartlett‟s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett‟s t -test. Where the data did not meet the homogeneity of variance, Student's t-test was performed to calculate significance (Gad, S.C. and Weil, C.S., 1994).JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 20 of 85



### 8. RESULTS

8 .1 Chemical Analysis (Dose Formulation Analysis) (Refer Appendix 7)

At the initiation of treatment period, the mean percentage recovery obtained for sodium 3- nitrobenzenesulfonate at 10, 100 and 1000 mg/kg b. wt./day we re 98.94, 98. 98 and 98.86%, respectively.

The analysis result revealed that the mean calculated concentration was within the acceptance

level of ± 10% of nominal concentration which indicates that the dose formulation of sodium 3- nitrobenzenesulfonate in reverse osmosis water was homogeneous and the concentration was within the acceptance level.

8 .2 Mortalities (Refer Table 1; Appendix 1)

No mortality was observed during treatment period in groups given reverse osmosis water and

animals treated with sodium 3-nitrobenzenesulfonate at 1 0, 100 and 10 00 mg/kg b. wt./day.

8.3 Clinical Observations (Refer Table 2; Appendix 2)

All the animals given reverse osmosis water and treated with 1 0, 100 and 1000 mg/kg b. wt./day of sodium 3-nitrobenzenesulfonate were normal throughout study period.

8.4 Body Weight (Refer Tables 3 and 4; Appendices 3 and 4)

No change in mean body weight and percent body weight change was observed in male rats

treated with sodium 3-nitrobenzenesulfonate at 1 0, 100 and 10 00 mg/kg b. wt./day when compared with the control group.

Statistically significant change in body weight change was observed during treatment period 1-11 and 1-TS in female rats treated with sodium 3-nitrobenzenesulfonate at 10 mg/kg b. wt./day when compared with the control group. However, this change is considered as non-treatment related because it lacks dose dependency and subsequent change in feed consumption.

8.5 F ee d Consumption (Refer Table 5; Appendix 5)

No change in f

ee d consumption was observed in rats treated with sodium 3-nitrobenzenesulfonate

at 1 0, 100 and 10 00 mg/kg b. wt./day when compared with the control group.JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 21 of 85

8.6 Organ Weight
Statistically significant reduction in testes weight was observed in animal treated with 100 mg/kg b. wt./day sodium 3-nitrobenzenesulfonate. However, such changes was not observed at 1000 mg/kg b. wt./day of sodium 3-nitrobenzensulfonate hence, change is considered as incidental in

nature

.

No treatment related changes were observed in absolute organ weight and relative organ weight

of rats treated with sodium 3-nitrobenzenesulfonate at 10, 100 and 1000 mg/kg b. wt./day when compared with the control group (Refer Appendix 6).

8.7 Macroscopic Pathological Findings
External and internal examination of terminally sacrificed rats did not reveal any gross lesions of pathological significance (Refer Appendix 6).JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 22 of 85



### 9. CONCLUSION

Based on the results of this study, it is concluded that sodium 3-nitrobenzensulfonate did not produce any adverse effect up to the dose level of 1000 mg/kg b. wt./day when administered through oral gavage for 14 consecutive days in Wistar rats under the conditions and procedures followed in the present study.

Based on the result of this dose range finding study, following dose levels are proposed for definitive study:

Dose L evels Proposed Dose Level

- I Proposed Dose Level

- I I

Low 10 62.5

Mid 100 250

High 1000 1000JRF Study Number: 410 -1- 04

- 5132 (Final Report) Page 23 of 85



### 10. REFERENCES

A. Shrimali , 2012: JRF Study N° 228

- 2

- 13

- 5312, “ Validation o f Analyti cal Method f or
Determination o f Sodium 3

- nitrobenzensulfonate Active Ingredient Concentration i n Dose

Formulation ”.

D. Ujawane, 2012: JRF Study N° 401

- 1

- 04

- 5131, “ Acute Oral Toxicity Study o f Sodium 3

-

n itrobenzensulfonate i n Rats ”.

Gad, S.C. and Weil, C.S. , 1994: “Statistics for Toxicologists”. In: Principles and Methods of Toxicology, 3 rd edition, Hayes A.W. (Ed), Raven P ress Ltd., New York, p p. 221

- 274.

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).

Organization for Economic Co

- operation and Development (OECD) for Testing of Chemicals, N° 407, entitled “Repeated Dose 28

- Day Oral Toxicity Study in Rodents”, adopted by the Council
on October 03, 2008.

The United States Environmental Protection Agency ( July 200 0 ). Health Effects Test Guidelines,

OPPTS 870. 3050 , EPA 712

- C

- 00

- 366, “Repeated Dose 28

- Day Oral Toxicity Study in Rodents”.JRF Study Number: 410-1-04-5132 (Final Report) Page 24 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

TABLE 1

Mortality Record

Group Dose

( m g /kg b. wt. /day ) Mortality % Mortality

Male (N=05) Female

(N=05) Total

(N=10) Mal e Female Total

G1 0 0 0 0 0 0 0

G2 10 0 0 0 0 0 0

G3 1 00 0 0 0 0 0 0

G4 1000 0 0 0 0 0 0

Key : N = Number of animalsJRF Study Number: 410-1-04-5132 (Final Report) Page 25 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

TABLE 2

Clinical Signs

–

Summary by Group



### Dose: G1-0; G2-10; G3-100; G4-1000 mg/kg b. wt./day

Male Number of Normal Animals during Week

1 2

G1 (N=05) 05 05

G2 (N=05) 05 05

G3 (N=05) 05 05

G4 (N=05) 05 05

Female
G1 (N=05) 05 05

G2 (N=05) 05 05

G3 (N=05) 05 05

G4 (N=05) 05 05

Key : N = Number of animalsJRF Study Number: 410-1-04-5132 (Final Report) Page 26 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

TABLE 3

Body Weight (g)

–

Group Mean Values

Dose: G1-0; G2-10; G3-1 00 ; G4-1000 mg /kg b. wt./day Sex: Male

Day G1 G2 G3 G4

Mean SD N Mean SD N Mean SD N Mean SD N

1 166.38 13.19 5 162.22 7.80 5 160.68 3.93 5 160.62 4.15 5

4 184.80 13.97 5 178.90 7.08 5 179.28 5.04 5 179.42 4.45 5

8 207.20 12.05 5 203.32 8.94 5 206.56 8.13 5 203.48 6.90 5

11 224.42 12.53 5 220.66 9.78 5 222.36 12.00 5 222.38 7.04 5

TS 246.54 13.32 5 241.76 11.02 5 244.82 12.86 5 244.04 7.24 5

Dose: G1-0; G2-10; G3-1 00 ; G4-1000 mg /kg b. wt./day Sex: Female

Day G1 G2 G3 G4

Mean SD N Mean SD N Mean SD N Mean SD N

1 129.52 4.80 5 131.46 5.46 5 129.10 4.41 5 132.00 4.41 5

4 141.02 8.84 5 139.54 7.55 5 139.66 6.49 5 140.38 6.01 5

8 153.22 10.34 5 148.14 7.55 5 150.82 6.96 5 149.64 7.24 5

11 165.50 12.21 5 154.94 7.87 5 159.84 9.89 5 158.20 6.22 5

TS 173.28 13.78 5 162.76 8.72 5 168.88 9.33 5 164.84 7.33 5

Keys: SD = Standard deviation, N = Number of animals, TS = Terminal sacrificeJRF Study Number: 410-1-04-5132 (Final Report) Page 27 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

TABLE 4

Mean Body Weight Change (%)

– Group Mean Values

Dose: G1-0; G2-10; G3-1 00 ; G4-1000 mg /kg b. wt./day Sex: Male

Day G1 G2 G3 G4

Mean SD N Mean SD N Mean SD N Mean SD N

1

- 4 11.10 1.34 5 10.32 1.25 5 11.57 1.23 5 11.71 0.95 5

1

- 8 24.69 2.68 5 25.39 3 .33 5 28.52 2.41 5 26.66 1.20 5

1

- 11 35.08 3.70 5 36.09 4.18 5 38.32 4.82 5 38.44 1.26 5

1

- TS 48.43 5.08 5 49.12 5.31 5 52.31 5.54 5 51.93 0.92 5

Dose: G1-0; G2-10; G3-1 00 ; G4-1000 mg /kg b. wt./day Sex: Female

Day G1 G2 G3 G4

Mean SD N Mean SD N Mean SD N Mean SD N

1

- 4 8.82 3.52 5 6.12 2.56 5 8.19 3.73 5 6.33 1.12 5

1

- 8 18.20 3.81 5 12.69 3.33 5 16.83 3.85 5 13.34 2.78 5

1

- 11 27.64 5.01 5 17.92  5.54 5 23.80 6.07 5 19.84 2.22 5

1

- TS 33.63 6.14 5 23.87  6.26 5 30.84 6.18 5 24.88 3.78 5

Keys: SD = Standard deviation, N = Number of animals, TS = Terminal sacrifice  - Significantly lower than control (p  0.05)JRF Study Number: 410-1-04-5132 (Final Report) Page 28 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

TABLE 5

F ee d Consumption (g/rat/day)

– Group Mean Values

Dose: G1-0; G2-10; G3-1 00 ; G4-1000 mg /kg b. wt./day Sex: Male

Day G1 G2 G3 G4

Mean SD n Mean SD n Mean SD n Mean SD n

1

- 4 19.91 1.16 2 19.09 0.02 2 20.75 1.77 2 19.50 1.16 2

4

- 8 20.66 0.00 2 20 .79 1.00 2 22.52 2.07 2 21.26 1.53 2

8

- 1 1 21.45 0.30 2 21.27 0.44 2 22.80 2.27 2 23.01 0.88 2

11

- TS 29.96 0.40 2 29.76 1.48 2 30.71 2.30 2 30.41 1.29 2

Dose: G1-0; G2-10; G3-1 00 ; G4-1000 mg /kg b. wt./day Sex: Female

Day G1 G2 G3 G4

Mean SD n M ean SD n Mean SD n Mean SD n

1

- 4 15.04 2.14 2 14.89 0.86 2 15.60 2.41 2 14.87 1.78 2

4

- 8 15.44 1.76 2 14.82 0.51 2 16.19 2.12 2 14.88 1.21 2

8

- 11 16.49 1.74 2 14.90 0.00 2 16.48 2.33 2 15.41 0.60 2

11

- TS 20.91 2.23 2 20.57 0.26 2 21.78 1.99 2 20.8 2 1.56 2

Keys: SD = Standard deviation, n = Number of cages, TS = Terminal sacrificeJRF Study Number: 410-1-04-5132 (Final Report) Page 29 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

FIGURE 1

Mean Body Weight (g) G1 to G4 - MaleJRF Study Number: 410-1-04-5132 (Final Report) Page 30 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

FIGURE 2

Mean Body Weight (g) G1 to G4

– FemaleJRF Study Number: 410-1-04-5132 (Final Report) Page 31 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

FIGURE 3

Mean Body Weight Change (%) (G1 to G4 )

– MaleJRF Study Number: 410-1-04-5132 (Final Report) Page 32 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

FIGURE 4

Mean Body Weight Change (%) (G1 to G4 ) - FemaleJRF Study Number: 410-1-04-5132 (Final Report) Page 33 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

FIGURE 5

Mean F ee d Consumption (g/rat/day) G1 to G4 - MaleJRF Study Number: 410-1-04-5132 (Final Report) Page 34 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

FIGURE 6

Mean F ee d Consumption (g/rat/day) G1 to G4- FemaleJRF Study Number: 410-1-04-5132 (Final Report) Page 35 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 1

Record of Animal Fate and Disposition

Group: G1; Dose: 0 mg/kg b. wt./day

Rat N° Sex Fate Day Rat N° Sex Fate Da y

1

2

3

4

5 M

M

M

M

M TS

TS

TS

TS

TS 15

15

15

15

15 6

7

8

9

10 F

F

F

F

F TS

TS

TS

TS

TS 15

15

15

15

15

Group: G2; Dose: 10 mg/kg b. wt./day

Rat N° Sex Fate Day Rat N° Sex Fate Day

1 1

1 2

1 3

1 4

1 5 M

M

M

M

M TS

TS

TS

TS

TS 15

15

15

15

15 16

17

18

19

20 F

F

F

F

F TS

TS

TS

TS

TS 15

15

15

15

15

Key: TS = Terminal sacrificeJRF Study Number: 410-1-04-5132 (Final Report) Page 36 of 85

APPENDIX 1 (Continued)

Group: G3; Dose: 100 mg/kg b. wt./day

Rat N° Sex Fate Day Rat N° Sex Fate Day

2 1

2 2

2 3

2 4

2 5 M

M

M

M

M TS

TS

TS

TS

TS 15

15

15

15

15 26

27

28

29

30 F

F

F

F

F TS

T S

TS

TS

TS 15

15

15

15

15

Group: G4; Dose: 10 00 mg/kg b. wt./day

Rat N° Sex Fate Day Rat N° Sex Fate Day

3 1

3 2

3 3

3 4

3 5 M

M

M

M

M TS

TS

TS

TS

TS 15

15

15

15

15 36

37

38

39

40 F

F

F

F

F TS

TS

TS

TS

TS 15

15

15

15

15

Key: TS = Terminal sacrificeJRF Study Number: 410-1-04-5132 (Final Report) Page 37 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 2



### Clinical Signs of Individual Rat

Group: G1; Dose: 0 mg/kg b. wt./day

Male On Experimental Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1

4 1 1 1 1 1 1 1 1 1 1 1 1 1 1

5 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Female On Experimental Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

6 1 1 1 1 1 1 1 1 1 1 1 1 1 1

7 1 1 1 1 1 1 1 1 1 1 1 1 1 1

8 1 1 1 1 1 1 1 1 1 1 1 1 1 1

9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

10 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Key: 1 = NormalJRF Study Number: 410-1-04-5132 (Final Report) Page 38 of 85

APPENDIX 2 (Continued)

Group: G2; Dose: 10 mg/kg b. wt./day

Male On Experimental Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Female On Experimental Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Key: 1 = NormalJRF Study Number: 410-1-04-5132 (Final Report) Page 39 of 85

APPENDIX 2 (Continued)

Group: G3; Dose: 100 mg/kg b. wt./day

Male On Experimental Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Female On Experimental Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

2 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Key: 1 = NormalJRF Study Number: 410-1-04-5132 (Final Report) Page 40 of 85

APPENDIX 2 (Continued)

Group: G4; Dose: 10 00 mg/kg b. wt./day

Male On Experimental Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Female On Experimen tal Day

Rat N° 1 2 3 4 5 6 7 8 9 10 11 12 13 14

3 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1

4 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Key: 1 = NormalJRF Study Number: 410-1-04-5132 (Final Report) Page 41 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 3

Body Weight (g) of Individual Rat

Group: G1; Dose: 0 mg/kg b. wt./day

Rat N° and Sex Experimental Day

1 4 8 11 TS

1M 189.9 209.6 228.4 245 .5 267.6

2M 158.8 179.6 200.0 212.8 231.8

3M 160.6 178.3 203.9 221.5 242.1

4M 161.9 180.5 204.2 224.2 249.3

5M 160.7 176.0 199.5 218.1 241.9

6F 134.9 154.6 167.5 180.0 188.5

7F 132.7 142.6 157.1 168.8 177.0

8F 130.8 141.6 155.2 172.4 181.9

9F 123.4 134.1 142.2 149.8 154.0

10F 125.8 132.2 144.1 156.5 165.0

Group: G2; Dose: 10 mg/kg b. wt./day

Rat N° and Sex Experimental Day

1 4 8 11 TS

11M 174.9 190.0 213.0 229.4 249.4

12M 163.8 179.6 207.2 226.7 251.2

13M 160.3 179.4 207.6 226.0 246.6

14M 1 56.3 173.4 198.5 215.0 236.9

15M 155.8 172.1 190.3 206.2 224.7

16F 140.7 150.6 159.3 162.9 170.6

17F 131.8 143.8 150.1 157.4 166.8

18F 127.3 136.9 148.8 154.2 161.8

19F 129.7 133.6 139.8 142.0 148.2

20F 127.8 132.8 142.7 158.2 166.4JRF Study Number: 410-1-04-5132 (Final Report) Page 42 of 85

APPENDIX 3 (Continued)

Group: G3; Dose: 100 mg/kg b. wt./day

Rat N° and Sex Experimental Day

1 4 8 11 TS

21M 166.6 185.9 216.7 234.5 254.4

22M 161.6 179.8 209.1 227.6 252.7

23M 159.6 181.4 209.1 227.3 250.9

24M 159.8 176.9 203.0 219.2 242.9

25M 155.8 172.4 194.9 203.2 223.2

26F 131.6 147.5 159.6 173.1 183.1

27F 131.8 145.8 156.8 167.8 173.8

28F 133.3 136.4 147.9 154.0 162.5

29F 123.3 133.6 143.8 152.2 162.6

30F 125.5 135.0 146.0 152.1 162.4

Group: G4; Dose: 10 00 mg/kg b. wt./day

Rat N° and Sex Experimental Day

1 4 8 11 TS

31M 166.6 186.1 212.5 231.3 254.5

32M 162.6 179.5 208.5 225.6 246.1

33M 158.6 179.3 200.5 220.8 242.0

34M 159.6 178.6 200.6 222.1 243.1

35M 155.7 173.6 195.3 212.1 234.5

36F 139.1 149.6 160.6 165.8 172.1

37F 131.4 140.9 153.2 162.0 171.6

38F 132.5 141.0 145.2 159.5 165.6

39F 129.6 136.8 146.4 152.0 155.6

40F 127.4 133.6 142.8 151.7 159.3JRF Study Number: 410-1-04-5132 (Final Report) Page 43 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 4

Mean Body Weight Change (%) of Individual Rat

Group: G1; Dose: 0 mg/kg b. wt./day

Rat N° & Sex During days Rat N° & Sex During days

1

- 4 1

- 8 1

- 11 1

- TS 1

- 4 1

- 8 1

- 11 1

- TS

1M 10.37 20.27 29.28 40.92 6F 14.60 24.17 33.43 39.73

2M 13.10 25.94 34.01 45.97 7F 7.46 18.39 27.20 33.38

3M 11.02 26.96 37.92 50.75 8F 8.26 18.65 31.80 39.07

4M 11.49 26.13 38.48 53.98 9F 8.67 15.24 21.39 24.80

5M 9.52 24.14 35.72 50.53 10F 5.09 14.55 24.40 31.16

Group: G2; Dose: 10 mg/kg b. wt./day

Rat N° & Sex During d ays Rat N° & Sex During days

1

- 4 1

- 8 1

- 11 1

- TS 1

- 4 1

- 8 1

- 11 1

- TS

11M 8.63 21.78 31.16 42.60 16F 7.04 13.22 15.78 21.25

12M 9.65 26.50 38.40 53.36 17F 9.10 13.88 19.42 26.56

13M 11.92 29.51 40.99 53.84 18F 7.54 16.89 21.13 27.10

14M 10. 94 27.00 37.56 51.57 19F 3.01 7.79 9.48 14.26

15M 10.46 22.14 32.35 44.22 20F 3.91 11.66 23.79 30.20JRF Study Number: 410-1-04-5132 (Final Report) Page 44 of 85

APPENDIX 4 (Continued)

Group: G3; Dose: 100 mg/kg b. wt./day

Rat N° & Sex During days Rat N° & Sex During days

1

- 4 1

- 8 1

- 11 1

- TS 1

- 4 1

- 8 1

- 11 1

- TS

21M 11.58 30.07 40.76 52.70 26F 12.08 21.28 31.53 39.13

22M 11.26 29.39 40.84 56.37 27F 10.62 18.97 27.31 31.87

23M 13.66 31.02 42.42 57.21 28F 2.33 10.95 15.53 21.91

24M 10.70 27.03 37.17 52.00 29F 8.35 16.63 23.44 31.87

25M 10.65 25.1 0 30.42 43.26 30F 7.57 16.33 21.20 29.40

Group: G4; Dose: 10 00 mg/kg b. wt./day

Rat N° & Sex During days Rat N° & Sex During days

1

- 4 1

- 8 1

- 11 1

- TS 1

- 4 1

- 8 1

- 11 1

- TS

31M 11.70 27.55 38.84 52.76 36F 7.55 15.46 19.19 23.72

32M 10.39 2 8.23 38.75 51.35 37F 7.23 16.59 23.29 30.59

33M 13.05 26.42 39.22 52.59 38F 6.42 9.58 20.38 24.98

34M 11.90 25.69 39.16 52.32 39F 5.56 12.96 17.28 20.06

35M 11.50 25.43 36.22 50.61 40F 4.87 12.09 19.07 25.04JRF Study Number: 410-1-04-5132 (Final Report) Page 45 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 5

F ee d Consumption (g/rat/day) of Individual Rat

Group: G1; Dose: 0 mg/kg b. wt./day

Cage N° & Sex During day s Cage N° & Sex During day s

1

- 4 4

- 8 8

- 11 11

- TS 1

- 4 4

- 8 8

- 11 11

- TS

1 M 20.73 20.65 21.23 29.68 3 F 16.55 16.68 17.72 22.48

2M 19.09 20.66 21.66 30.24 4 F 13.53 14.19 15.26 19.33

Group: G2; Dose: 10 mg/kg b. wt./day

Cage N° & Sex During day s Cage N° & Sex During day s

1

- 4 4

- 8 8

- 11 11

- TS 1

- 4 4

- 8 8

- 11 11

- TS

5 M 19.07 21.50 21.58 30.80 7 F 15.50 14.46 14.90 20.75

6M 19.10 20.08 20.96 28.71 8 F 14.28 15.18 14.89 20.38

Group: G3; Dose: 100 mg/kg b. wt./day

Cage N° & Sex During day s Cage N° & Sex During day s

1

- 4 4

- 8 8

- 11 11

- TS 1

- 4 4

- 8 8

- 11 11

- TS

9 M 22.00 23.98 24.40 32.33 11 F 17.30 17.69 18.13 23.18

10M 19.49 21.05 21.19 29.08 12 F 13.89 14.69 14.83 20.37

Group: G4; Dose: 10 00 mg/kg b. wt./day

Cage N° & Sex During day s Cage N° & Sex During day s

1

- 4 4

- 8 8

- 11 11

- TS 1

- 4 4

- 8 8

- 11 11

- TS

13 M 20.32 22.34 23.63 31.32 15 F 16.13 15.73 15.83 21.92

14M 18.68 20.17 22.38 29.50 16 F 13.61 14.02 14.98 19.72JRF Study Number: 410-1-04-5132 (Final Report) Page 47 of 85

APPENDIX 6 (Continued)



### PERSONNEL INVOLVED

Study Pathologist : DR. ARABINDA ADAK, M.V.Sc.

Study Personnel : BHAVNA PRAJAPATI, B.Sc., D.M.L.T.
PRAVINA PATEL, B.S c., D.M.L.T.
SNEHA VINODAN, M.Sc. M.L.T.

Statistician : RAJESH PATE L, B.Sc. (Math)JRF Study Number: 410-1-04-5132 (Final Report) Page 48 of 85

APPENDIX 6 (Continued)

CONTENTS

Page N°

TITLE PAGE ............................................................................................................................................. 46

PERSONNEL INVOLVED ........................................................................................................................ 47

CONTENTS ................................................................................................................................................ 48

LIST OF ABBREVIATIONS ..................................................................................................................... 49

1. EXPERIMENTAL PROCEDURE ................................................................................................ 50

2. RESULTS AND DISCUSSION .................................................................................................... 50 2.1 Terminal Body Weight (g), Absolute Organ Weight (g) and Organ Weight Relative to Terminal Body Weight (%) ............................................................................................... 50 2.2 Gross Findings ................................................................................................................. 50

TABLES

A. Terminal Body Weight, Absolute Organ Weight (g) and Organ Weight Relative to Terminal Body Weight (%) - Group Mean Values ........................................................... 51

B. Gross Findings- Summary by Groups .............................................................................. 51

APPENDICES A. Terminal Body Weight (g)

, Absolute Organ Weight (g) and Organ Weight Relative to Terminal Body Weight (%) ............................................................................................... 52 B. Gross Findings of Individual Rat ...................................................................................... 54JRF Study Number: 410-1-04-5132 (Final Report) Page 49 of 85

APPENDIX 6 (Continued)

LIST OF ABBREVIATIONS

% - Percentage b.wt. - Body Weight

F

- Female

g

- Gram

M

- Male

G1

- Control ( 0 mg /kg b.wt. /day )

G2

- Low D ose G roup ( 1 0 mg /kg b.wt. /day )

G3

- Mid D ose G roup ( 100 mg /kg b.wt. /day )

G4

- High Dose Group ( 1000 mg /kg b.wt. /day ) kg

- Kilogram

mg

- Milligram

- Milliliter

NAD

- No A bnormality D etected

N

- Number of A nimals

N°

- Number

SD

- Standard D eviationJRF Study Number: 410-1-04-5132 (Final Report) Page 50 of 85

APPENDIX 6 (Continued)

1. EXPERIMENTAL PROCEDURE

During terminal sacrifice, all the rats belonging to all the groups (G1 to G4 ) were euthanised by carbon dioxide asphyxiation and subjected to full gross necropsy under the direct supervision of a veterinary pathologist. First the carcasses were examined for external abnormalities followed by examination of organs by cut opening the thoracic and abdominal cavities. Testes and epididymes from male rats were weighed and relative weights (organ weight relative to terminal body weight)

were calculated.

2. RESULTS AND DISCUSSION

2.1 Terminal Body Weight (g), Absolute Organ Weight (g) and Organ Weight Relative to Terminal Body Weight (%) [Tables A and Appendices A ]
Terminal body weight and organ weight (both absolute and relative) of testes and epididymes of male animals belonging to group G2 to G4 was comparable to the control group (G1). However,

st atistically significant decrease in absolute weight of testes was observed in male animals at 100

mg /kg b. wt./day when compared to the control group (G1). This effect was considered incidental and not treatment related as there was no dose response relationship and the effect was of low magnitude.

2.2 Macroscopic Findings [Table B and Appendix B ]

External External examination of the terminally sacrificed animals of all the groups (G1 to G4) did not reveal any lesion of pathological significance.

Internal Internal examination of the terminally sacrificed animals of all the groups (G1 to G4) did not reveal any lesion of pathological significance.JRF Study Number: 410-1-04-5132 (Final Report) Page 51 of 85

APPENDIX 6 (Continued)

TABLE A: Terminal Body Weight (g), Absolute Organ Weight (g) and Organ Weight Relative to
Terminal Body Weight (%)

– Group Mean Values

Parameter s Absolute organ weight (g) Organ weight r elative to terminal body weight (%)
Group G1 G2 G3 G4 G1 G2 G3 G4

Dose (mg/kg b. wt./day) 0 10 100 1000 0 10 100 1000

N 5 5 5 5 5 5 5 5

Terminal Body Weight Mea n 246.540 241.760 244.820 244.040 Not applicable

SD 13.320 11.018 12.864 7.241 Not applicable

Testes Mean 3.080 3.024 2.820  3.080 1.250 1.252 1.156 1.263

SD 0.175 0.139 0.096 0.097 0.043 0.066 0.098 0.058

Epididymes Mean 0.601 0.561 0.554 0.548 0.2 45 0.233 0.228 0.225

SD 0.105 0.042 0.116 0.027 0.051 0.024 0.054 0.013

TABLE B: Gross Findings

– Summary by Groups

Group & Dose G1 0 mg/kg b. wt./day G2

10 mg/kg b. wt./day G3

100 mg/kg b. wt./day G4

10 00 mg/kg b. wt./day
Organs & Lesions Sex M F M F M F M F

N° of Animals 5 5 5 5 5 5 5 5

External No Abnormality Detected 5 5 5 5 5 5 5 5

Internal No Abnormality Detected 5 5 5 5 5 5 5 5JRF Study Number: 410-1-04-5132 (Final Report) Page 52 of 85

APPENDIX 6 (Continued)

APPENDIX A: Terminal Body Weight (g), Absolute Organ Weight (g) and Organ Weight Relative to Terminal Body Weight (%) of Individual Rat

Group: G1; Dose: 0 mg /kg b. wt./day Sex: Male

Rat N° Terminal b. wt. Absolute organ weight (g) Organ weight relative to terminal body weight (%)
Testes Epididymes Testes Epididymes

1 267.6 3.296 0.530 1.232 0.198

2 23 1.8 2.986 0.746 1.288 0.322

3 242.1 2.882 0.503 1.19 0.208

4 249.3 3.229 0.676 1.295 0.271

5 241.9 3.008 0.550 1.243 0.227

Group: G2; Dose: 10 mg /kg b. wt./day Sex: Male

Rat N° Terminal b. wt. Absolute weight (g) Organ weight relative to terminal bod y weight (%)
Testes Epididymes Testes Epididymes

11 249.4 2.925 0.596 1.173 0.239

12 251.2 3.111 0.518 1.238 0.206

13 246.6 3.214 0.577 1.303 0.234

14 236.9 2.871 0.514 1.212 0.217

15 224.7 2.999 0.602 1.335 0.268JRF Study Number: 410-1-04-5132 (Final Report) Page 53 of 85

APPENDIX 6 (Continued)

APPENDIX A (Continued)

Group: G3; Dose: 100 mg /kg b. wt./day Sex: Male

Rat N° Terminal b. wt. Absolute weight (g) Organ weight relative to terminal body weight (%)
Testes Epididymes Testes Epididymes

21 254.4 2.787 0.506 1.096 0.199

22 252.7 2.673 0.433 1.058 0.171

23 250.9 2.8 48 0.509 1.135 0.203

24 242.9 2.863 0.738 1.179 0.304

25 223.2 2.929 0.582 1.312 0.261

Group: G4; Dose: 10 00 mg /kg b. wt./day Sex: Male

Rat N° Terminal b. wt. Absolute weight (g) Organ weight relative to terminal body weight (%)
Testes Epididymes T estes Epididymes

31 254.5 3.118 0.535 1.225 0.21

32 246.1 3.118 0.542 1.267 0.22

33 242.0 2.926 0.595 1.209 0.246

34 243.1 3.056 0.534 1.257 0.22

35 234.5 3.181 0.533 1.357 0.227JRF Study Number: 410-1-04-5132 (Final Report) Page 54 of 85

APPENDIX 6 (Continued)

APPENDIX B : Gross Findings of Individual Rat

Group: G1; Dose : 0 mg /kg b. wt./day Sex: Male

Rat N° Mode

of Death Gross Findings

External Internal

1 TS NAD NAD

2 TS NAD NAD

3 TS NAD NAD

4 TS NAD NAD

5 TS NAD NAD

Group: G1; Dose: 0 mg/kg b. wt./day Sex: Female

Rat N° Mode

of Death Gross Findings

External Interna l

6 TS NAD NAD

7 TS NAD NAD

8 TS NAD NAD

9 TS NAD NAD

10 TS NAD NADJRF Study Number: 410-1-04-5132 (Final Report) Page 55 of 85

APPENDIX 6 (Continued)

APPENDIX B (Continued)

Group: G2; Dose: 10 mg/kg b. wt./day Sex: Male

Rat N° Mode

of Death Gross Findings

External Internal

1 1 TS NAD NAD

1 2 TS NAD NAD

1 3 TS NAD NAD

14 TS NAD NAD

15 TS NAD NAD

Group: G2; Dose: 10 mg/kg b. wt./day Sex: Female

Rat N° Mode

of Death Gross Findings

External Internal

1 6 TS NAD NAD

1 7 TS NAD NAD

1 8 TS NAD NAD

19 TS NAD NAD

20 TS NAD NADJRF Study Number: 410-1-04-5132 (Final Report) Page 56 of 85

APPENDIX 6 (Continued)

APPENDIX B (Continued)

Group: G3; Dose: 100 mg/kg b. wt./day Sex: Male

Rat N° Mode

of Death Gross Findings

External Internal

21 TS NAD NAD

22 TS NAD NAD

23 TS NAD NAD

24 TS NAD NAD

25 TS NAD NAD

Group: G3; Dose: 100 mg/kg b. wt./day Sex: Female

Rat N° Mode

of Death Gross Findings

Extern al Internal

26 TS NAD NAD

27 TS NAD NAD

28 TS NAD NAD

29 TS NAD NAD

30 TS NAD NADJRF Study Number: 410-1-04-5132 (Final Report) Page 57 of 85

APPENDIX 6 (Continued)

APPENDIX B (Continued)

Group: G4; Dose: 10 00 mg/kg b. wt./day Sex: Male

Rat N° Mode

of Death Gross Findings

External Internal

31 TS NAD NAD

32 TS N AD NAD

33 TS NAD NAD

34 TS NAD NAD

35 TS NAD NAD

Group: G4; Dose: 10 00 mg/kg b. wt./day Sex: Female

Rat N° Mode

of Death Gross Findings

External Internal

36 TS NAD NAD

37 TS NAD NAD

38 TS NAD NAD

39 TS NAD NAD

40 TS NAD NADJRF Study Number: 410-1-04-5132 (Final Report) Page 58 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 7

Dose Formulation ReportJRF Study Number: 410-1-04-5132 (Final Report) Page 59 of 85

APPENDIX 7 (Continued)JRF Study Number: 410-1-04-5132 (Final Report) Page 60 of 85

APPENDIX 7 (Continued)JRF Study Number: 410-1-04-5132 (Final Report) Page 61 of 85

APPENDIX 7 (Continued)JRF Study Number: 410-1-04-5132 (Final Report) Page 62 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 8

Analytical Report on Residues of Organochlorine and Organophosphorus Pesticides and Heavy Metals in F ee d, Water and Paddy Husk

A. Chemical Analysis Report of Rodent FeedJRF Study Number: 410-1-04-5132 (Final Report) Page 63 of 85

APPENDIX 8 (Continued)

B. Chemical Analysis Report of Paddy HuskJRF Study Number: 410-1-04-5132 (Final Report) Page 64 of 85

APPENDIX 8 (Continued)

C. Chemical Analysis Report of WaterJRF Study Number: 410-1-04-5132 (Final Report) Page 65 of 85



### Repeated Dose 14-Day Oral Toxicity Study o f Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 9

Results of Microbiological Analysis of Rat Pellet F ee d, Water and Paddy Husk

A. Report for Microbiological Analysis of Rodent DietJRF Study Number: 410-1-04-5132 (Final Report) Page 66 of 85

APPENDIX 9 (Continued)

B. Report for Microbiological Analysis of WaterJRF Study Number: 410-1-04-5132 (Final Report) Page 67 of 85

APPENDIX 9 (Continued)

B. Report for Microbiological Analysis of WaterJRF Study Number: 410-1-04-5132 (Final Report) Page 68 of 85

APPENDIX 9 (Continued)

C. Microbial Analysis Report of Bedding MaterialJRF Study Number: 410-1-04-5132 (Final Report) Page 69 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 10

Signed Study Plan

JRF Study Number: 410-1- 04 -5132 Page 1 of 14

STUDY PLAN

REPEATED DOSE 14-DAY ORAL TOXICITY STUDY OF

SODIUM 3-NITROBENZENSULFONATE IN WISTAR RATS

(DOSE RANGE FINDING STUDY)

GUIDELINES: U.S. EPA OPPTS 870.3050 and OECD N° 407

SPONSOR

TIARCO CHEMICAL

1300 TIARCO DRIVE DALTON, GA 30720

TECHNICAL CONTACT : JEFF VANDERLIP

REGULATORY CONTACT : BENNETT KING

STUDY DIRECTOR: DEEPAK UJAWANE

TEST FACILITY

JAI RESEARCH FOUNDATION

DEPARTMENT OF TOXICOLOGY

VALVADA

- 396 108
DIST. VALSAD

GUJARAT
INDIA

OCTOBER - 2012JRF Study Number: 410-1-04-5132 (Final Report) Page 70 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 2 of 14

CONTENTS PAGE N°

TITLE PAGE

................................ ................................ ................................ ................................ ................................ 1

CONTENTS

.................................................................................................................................................................. 2

1. GENERAL INFORMATION

.............................................................................................................................. 4 1.1 Study Professionals

................................................................................................................................ 4 1.2 Test Facility Management

..................................................................................................................... 4

1.3 Technical Contact

.................................................................................................................................. 4 1.4 Regulatory Contact

................................................................................................................................ 4 1.5 Study Schedule

...................................................................................................................................... 4 1.6 Study Plan and Amendment (if any) Distribution

.................................................................................. 5

2. INTRODUCTION

............................................................................................................................................... 5 2.1 Objective

................................................................................................................................................ 5 2.2 Regulatory Guidelines

........................................................................................................................... 5 2.3 Test Method Principle

............................................................................................................................ 5

2.4 Test Item

................................................................................................................................................ 5

3. GOOD LABORATORY PRACTICE (GLP)

..................................................................................................... 6 3.1 GLP Compliance

................................................................................................................................... 6 3.2 Standard Operating Procedures (SOP)

................................................................................................... 6 3.3 Amendment to Study Plan

..................................................................................................................... 6 3.4 Deviation from the Study Plan

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

.................................................................................................................................. 7

4. ANIMAL WELFARE

......................................................................................................................................... 7 4.1 Humane Endpoint

.................................................................................................................................. 7

5. TEST SYSTEM

................................................................................................................................................... 7 5.1 Test System Specification

...................................................................................................................... 7 5.2 Justification of Test System

................................................................................................................... 7 5.3 Location of the Study

............................................................................................................................. 8 5.4 Experimental Room Sanitation

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

5.5 Acclimatization

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

5.6 Environmental Conditions

..................................................................................................................... 8 5.7 Housing

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

5.8 Diet and Water

....................................................................................................................................... 8 5.9 Allocation to Groups

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

5.10 Animal Identification

............................................................................................................................. 9JRF Study Number: 410-1-04-5132 (Final Report) Page 71 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 3 of 14

CONTENTS (Continued) PAGE N°

6. EXPERIMENTAL PROCEDURE

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

6.1 Vehicle and Justification for Choice

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

6.2 Route and Mode of Administration and Justification for Choice

.......................................................... 9
6.3 Test Formulation Preparation

................................................................................................................ 9
6.4 Dosing

.................................................................................................................................................... 9
6.5 Method of Chemical Analysis

............................................................................................................. 10
6.6 Duration of Dosing

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

6.7 Dose Levels and Dose Justification

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

7. OBSERVATIONS

............................................................................................................................................. 10
7.1 Clinical Signs

....................................................................................................................................... 10
7.2 Mortality and Morbidity

...................................................................................................................... 10
7.3 Body Weight

........................................................................................................................................ 10
7.4 Feed Consumption

............................................................................................................................... 11
7.5 Gross Pathological Examination

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

8. DATA AND REPORTING

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

8.1 Tabulation

............................................................................................................................................ 11
8.2 Statistical Analysis

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

9. REPORT

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

10. ARCHIVES

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

11. REFERENCES

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

12. STUDY PLAN APPROVAL

............................................................................................................................. 14JRF Study Number: 410-1-04-5132 (Final Report) Page 72 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 4 of 14

1. GENERAL INFORMATION

1.1 Study Professionals

Name of Personnel Qualification Profession Experience Training

Deepak Ujawane (Study Director) M.Pharm. Toxicologist 3 years Declared Study Director for repeated dose toxicity studies

Mukesh Posia M.V.Sc. Veterinarian 2 years Trained as study veterinarian

Arabinda Adak M.V.Sc. Pathologist 5 years Trained as a study Toxico

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

1.2 Test Facility Management : T. G. Marvania, Ph.D. Deputy Director 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)

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 1.5 Study Schedu le

Study Initiation Date : October 10, 2012 Experiment Start Date : October 12, 2012 Experiment Completion : Latest by November 2012 Draft Report Submission : Latest by November 2012 Study Completion : Within two weeks from the date of receipt of comments on the final draft report from the Sponsor.JRF Study Number: 410-1-04-5132 (Final Report) Page 73 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 5 of 14

1.6 Study Plan and Amendment (if any) Distribution

a) Original copy in Archive and Study Sponsor; b) Photocopy to Study Director c) Photocopy to Pathology and d) Photocopy to QAU.

2. INTRODUCTION

2.1 Objective
The objective of this study is to determine doses for Reproduction/Developmental Toxicity Screening Test

of sodium 3-nitrobenzensulfonate in Wistar Rats.

2.2 Regulatory Guidelines
This study will be conducted based on following guidelines with exception of duration of treatment and Clinical Pathology and Pathology:

The United States Environmental Protection Agency (July 2000). Health Effects Test Guidelines, OPPTS 870.3050, EPA 712-C- 00

- 366, “Repeated Dose 28- Day Oral Toxicity Study in Rodents”;

and
Guidelines of Organization for Economic Co-operation and Development (OECD) for Testing of Chemicals, N° 407, entitled “Repeated Dose 28

- Day Oral Toxicity Study in Rodents”, adopted by the Council on October 03, 2008.

2.3 Test Method Principle

The test item will be administered by oral gavage for at least 14 days to 3 groups of male and female rats. A concurrent control group of male and female rats will receive vehicle alone. During the experimental period, all the animals will be observed closely for signs of toxicity. All the animals will be humanely sacrificed and subjected to gross examination at the end of the treatment period.

2.4 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 : Sodium 3

- nitrobenzensulfonate

IUPAC Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CA Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CAS Number : 127

- 68

- 4 Analysed Concentration : 96.9% (In formation provided by the Sponsor via CoA)

Batch N° : 50623 Supplied by : Reservol LPJRF Study Number: 410-1-04-5132 (Final Report) Page 74 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 6 of 14

Manufactured by : Tiarco Chemical

Date of Manufacture : September 08, 2011 Date of Expiry : September 08, 2013 Appearance : 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

identifica tion 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 between 40 and 90 F (room temperature 15

- 30 C) 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

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 for Economic

Co -operation and Development, Paris (1998) and all subsequent OECD consensus documents.

3.2 Standard Operating Procedures (SOP)

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 be 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 De viation from the Study Plan

Any deviation (unplanned change) from the study plan will be documented in study file and reported in the study report.JRF Study Number: 410-1-04-5132 (Final Report) Page 75 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 7 of 14

3.5 Quality Assurance

This study plan has been verified by JRF QAU and documented (Number 48732). 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 and reporting of findings to the Study Director and the Test Facility Management will be incorporated in the study report.

4. ANIMAL WELFARE

The study will be undertaken in compliance with the „Guidelines for Laboratory Animals Facility‟ issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. These guidelines promote the humane care of animals used in research by providing specifications that will enhance animal well being and experimental quality for the advancement of biological knowledge

that is relevant to humans and animals.

Jai Research Foundation is committed to enhancing animal welfare and ensures that studies are designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with Jai Research Foundation‟s policy on animal welfare. Project proposal for the experimentation is subject to approval by Institutional Animal Ethics Committee (IAEC), Jai Research Foundation.

JRF is also accredited with AAALAC (Association for Assessment and Accreditation of Laboratory Animals Care) that promotes humane care of animals during the experiments.

4.1 Humane Endpoint

Moribund animals or animals obviously in pain or showing signs of severe and enduring distress shall be humanely sacrificed and are considered in the interpretation of the study results in the same way as animals that died in experiment.

5. TEST SYSTEM

5.1 Test System Specification

Healthy, young adult rats ( Rattus norvegicus ) of Wistar strain will be obtained from the Animal Breeding Facility, Jai Research Foundation for the use in study. Female rats will be nulliparous and non-pregnant. At the initiation of dosing rats will not be more then 9 weeks old.

5.2 Justification of Test System

Rat is selected as a test system because it is a readily available laboratory rodent species. This species has been historically shown to be a suitable model for repeated dose toxicity studies. The OECD, EPA and other regulatory authorities also recommend it. In addition, a large amount of background knowledge was

accumulated on this strain.JRF Study Number: 410-1-04-5132 (Final Report) Page 76 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 8 of 14

5.3 Location of the Study

During the study period, the experimental animals will be kept in the Barrier Maintained Rodent Facility; the experimental room number will be specified in the study schedule.

5.4 Experimental Room Sanitation
Each day floor and all worktops will be swept and mopped with a disinfectant solution.

5.5 Acclimatization

A larger number of animals than necessary for the study will be ordered to permit the selection and/or replacement of individuals before the start of the treatment. A total of 25 male and 25 female rats will be selected for acclimatization. After acclimatization, 20 males and 20 females will be selected for the experiment. Remaining 5 male and 5 female will be returned to Animal Breeding Facility, Jai Research Foundation.Animals will be received into the experimental room and acclimatized for a minimum period of 5 days prior to the start of dosing. During acclimatization period, all animals will be observed daily for clinical signs at least once. During acclimatization, animals will be marked with temporary identification number on the base of the tail using marker pen.

5.6 Environmental Conditions
Rats will be maintained in an environment controlled room at a temperature of 22 ± 3 °C and relative humidity of 30 to 70 percent. The photoperiod will be 12 hour light and 12 hour darkness, light hours being 06.00 - 18.00 hour. Light period will be extended appropriately whenever required for practical feasibility of study related work. Air changes will be minimum 15 per hour.

5.7 Housing

Animals will be housed in groups of 2 or 3 rats/cage/sex during acclimatization and treatment period in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage will be fitted with a stainless steel top grill having provision for keeping rat pellet feed and a polypropylene water bottle with stainless steel drinking nozzle. Cages will be placed on 5/6 tier racks and cage rotation will be done atleast at weekly interval. Cages will be changed atleast twice a week. The bottom of the cages will be layered with clean sterilized rice (paddy) husk as the bedding. Contaminant analysis of samples of the bedding material is being performed at every six months intervals at Jai Research Foundation and most recent results will be included in the final report.

5.8 Diet and Water
The experimental animals will be fed with standard rodent pellet diet ad libitum with an unlimited supply of clean and filtered drinking water (filtered through Reverse Osmosis water filter system) in polypropylene bottles.JRF Study Number: 410-1-04-5132 (Final Report) Page 77 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 9 of 14

Quality of feed and water is being monitored regularly at Jai Research Foundation. The most recent result of microbial and chemical contaminant analysis will be incorporated in the final report.

5.9 Allocation to Groups

Prior to randomization, the animals will be weighed. Only healthy animals will be allocated to the study. Rats will be randomly allocated to different groups using an in-house developed and validated computer software program. Animals will be divided into four groups (G1-control, G2-low dose, G3-mid dose and

G4 -high dose). Each group comprised 5 males and 5 females. At the start of dosing, the body weight variation among the animals will be within ± 20% of the mean body weight for each sex.

5.10 Animal Identification

Each animal will be identified with unique number tattooed at the base of tail after randomization and each cage will have a colored cage label preferably showing study N°, study code, test item code, group number & sex, dose, cage number and animal number.

6. EXPERIMENTAL PROCEDURE

6.1 Vehicle and Justification for Choice

The reverse osmosis water (RO) is selected as a vehicle based on the solubility check performed at JRF and after consultation with the Sponsor.

6.2 Route and Mode of Administration and Justification for Choice

The route of test item administration will be through oral gavage since it will provide an exaggerated model of the reasonably foreseeable exposure in humans.

6.3 Test Formulation Preparation

The test item will be mixed with RO water to obtain the desired concentration. The test solutions will be prepared every day prior to dosing and will be used within 4 hours. Prior to dosing, the test solutions will be thoroughly mixed using magnetic stirrer and during dosing, the test solutions will be mixed intermittently in order to administer a homogeneous preparation.

6.4 Dosing

Test solutions will be administered once daily, by gavage, at approximately the same time each day, using intubation cannula attached to a graduated syringe. A constant dose volume of 10 mL/kg will be used and individual doses will be adjusted according to the most recently recorded body weight of animal. The control group animals will be given vehicle only.JRF Study Number: 410-1-04-5132 (Final Report) Page 78 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 10 of 14

6.5 Method of Chemical Analysis

The homogeneity and stability of test item in vehicle has been established in JRF Study N° 228-2- 13 -5312 following the validated method. The dose formulation will be analysed for active ingredient (a.i.) concentration and homogeneity at the initiation of the treatment period of the study. Details will be reported in the final report.

6.6 Duration of Dosing

The duration of dosing will be at least 14 days which is selected after consultation with Sponsor. The first day of dose administration will be designated as day 1 for each animal.

6.7 Dose Levels and Dose Justification

Based on results of acute oral toxicity study with same test item (JRF Study N° 401-1- 04 -5131), the following three dose levels are selected after consulting with Sponsor. Dose levels for study is (low dose - 10, mid dose - 100 and high dose -1000 mg/kg b. wt./day) of the test

item.

Table 1: Grouping and Animal Identification after Randomization

Group N° Group Dose (mg/kg b. wt./day) Number of

Animals Animal Identification

Number Ca ge Number

M F M F M F

G1 Control 0 5 5 1

- 5 6

- 10 1

- 2 3

- 4

G2 Low Dose 10 5 5 11

- 15 16

- 20 5

- 6 7

- 8

G3 Mid Dose 100 5 5 21

- 25 26

- 30 9

- 10 11

- 12

G4 High Dose 1000 5 5 31

- 35 36

- 40 13

- 14 15

- 16

7. OBSERVATIONS

Following observations will be recorded:

7.1 Cl inical Signs

All animals will be observed at least once a day, preferably at the same time each day.

7.2 Mortality and Morbidity

All animals will be checked at least twice a day for mortality and signs of morbidity. Moribund animals will be sacrificed humanely. All rats that die or are sacrificed will be subjected to post-mortem

examination.

7.3 Body Weight

Body weight will be recorded individually for all animals on the day of randomization, at the beginning of the treatment and at least twice weekly thereafter.JRF Study Number: 410-1-04-5132 (Final Report) Page 79 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 11 of 14

7.4 Feed Consumption

The feed consumption will be determined at least twice weekly and most preferably on same intervals as body weight recording.

7.5 Gross Pathological Examination

All animals will be euthanised by carbon dioxide asphyxiation and subjected to a full gross necropsy under the direct supervision of a veterinary pathologist at the end of the treatment period. Animals, which are found dead after regular working hours, will be refrigerated and necropsy will be performed at the start of the next working day.

All animals will be examined carefully for external abnormalities. The thoracic and abdominal cavities will be cut opened and a thorough examination of the organs will be carried out to detect abnormalities.

Organ weight will be recorded for the testes and epididymis for all male animals except on those found

dead or sacrificed moribund.

8. DATA AND REPORTING

8.1 Tabulation
All study observations will be recorded in standard formats.

8 .2 Statistical Analysis

Department of Bio-statistics and Systems Information, Jai Research Foundation will process the raw data to get group means and standard deviations with significance between the control and treated groups, using

in -house developed statistical software. The data will be summarised in tabular form. All the parameter characterised by continuous data such as body weight, feed consumption, organ weight will be subjected to Bartlett‟s test to meet the homogeneity of variance before conducti ng Analysis of Variance (ANOVA) and

Dunnett‟s t -test. Where the data do not meet the homogeneity of variance, Student's t-test will be performed to calculate significance (Gad, S.C. and Weil, C.S., 1994).

9. REPORT

Unless otherwise requested by the Sponsor, one copy of the final repot along with one soft copy in PDF will be issued to the Sponsor. The report will include, but not be limited to:

- The name and address of the Sponsor and the test facility, and the study dates
- Individual responsible for conduct and reporting of the study

- test item details
- The description of the test animals, including species strain used, source, number, sex, body weight range, age and method of identificationJRF Study Number: 410-1-04-5132 (Final Report) Page 80 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 12 of 14

- Test environment including temperature, humidity, air changes, cage condition and photoperiod
- Details of feed and water quality, analysis report of feed and water

Test procedure will include the following data:

- Method of randomization used
- Dose, route of administration and duration of treatment

The results will include the following data:
- Toxic response and other effects by sex and dose
- Mortality and survival
- Clinical signs of toxicity
- Death during the study or whether animals survived to termination
- Observation of each abnormal sign and its subsequent course
- Body weights and feed consumption data

- Relative and absolute organ weight data

- Necropsy findings

- Statistical treatment of results

- Discussion of results and conclusion

- Signed study plan and study plan amendment(s) (if any)

- Record of deviation(s) from the study plan (if any)

10. ARCHIVES

On completion of the study, all original raw data including any storage medium for electronically recorded data, documentation, the signed study plan, study plan amendment (if any), the draft report, a copy of the 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: 410-1-04-5132 (Final Report) Page 81 of 85
APPENDIX 10 (Continued)

JRF Study Number: 410-1- 04 -5132 Page 13 of 14

11. REFERENCES

A. Shrimali , 2012: JRF Study N° 228

- 2

- 13

- 531 2, “ Validation of Analytical Method for Determination of

Sodium 3

- nitrobenzensulfonate Active Ingredient Concentration in Dose Formulation ”.

D. Ujawane, 2012: JRF Study N° 401

- 1

- 04

- 5131, “Acute Oral Toxicity Study of Sodium 3

- nitrobenzensulfonate in Rats”.

Gad, S.C. and Weil, C.S., 1994: “Statistics for Toxicologists”. In: Principles and Methods of Toxicology,

3 rd edition, Hayes A.W. (Ed), Raven press Ltd., New York, p. 221

- 274.

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

Organization for Economic Co

- operation and Development (OECD) for Testing of Chemicals, N° 407, entitled “Repeated Dose 28

- Day Oral Toxicity St udy in Rodents”, adopted by the Council on October 03, 2008.

The United States Environmental Protection Agency (July 2000). Health Effects Test Guidelines, OPPTS
870.3050, EPA 712

- C

- 00

- 366, “Repeated Dose 28

- Day Oral Toxicity Study in Rodents”.JRF Study Number: 410-1-04-5132 (Final Report) Page 82 of 85
APPENDIX 10 (Continued)JRF Study Number: 410-1-04-5132 (Final Report) Page 83 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 11

Certificate of Analysis of Sodium 3-nitrobenzensulfonateJRF Study Number: 410-1-04-5132 (Final Report) Page 84 of 85



### Repeated Dose 14-Day Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)

APPENDIX 12

GLP Endorsement of ComplianceJRF Study Number: 410-1-04-5132 (Final Report) Page 85 of 85

APPENDIX 12 (Continued)