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 

CONTENTS .......................................................................................................................................... 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 ..................................................................................................16
JRF Study Number: 410-1-04-5132 (Final Report) Page 7 of 85

CONTENTS (Continued) 

PAGE N° 

5. EXPERIMENTAL PROCEDURE .........................................................................................17

 5.1 Vehicle 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. Clinical 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 ................................................ 28 
JRF 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. Feed 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 Feed, Water and Paddy Husk ...................................62

 9. Results of Microbiological Analysis of Rat Pellet Feed, Water and Paddy Husk.......65

10. Signed Study Plan .......................................................................................................69

 11. Certificate of Analysis of Sodium 3-nitrobenzensulfonate ......................................... 83 

 12. GLP Endorsement of Compliance ...............................................................................84

 

JRF 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 (Group 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 (1000 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 Deviation

TOX - Department of Toxicology

TS - Terminal Sacrifice

TICO - Test Item Control Office

TIDS - Test Item Data Sheet

% - Percent

°C - Degree Centigrade

- - Not Applicable
JRF 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 Guidelines of the OECD N° 407 (October 3, 2008).

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 (G4-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 feed 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 feed 

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-II

Low 10 62.5

Mid 100 250

High 1000 1000
JRF 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 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. 

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 : 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% (Refer Certificate of Analysis in Appendix N° 11)

Batch N° : 50623

Supplied by : Reservol LP

Manufactured By : Tiarco Chemical 

Date of Manufacture : September 08, 2011

Date of Expiry : 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 characterization of the test item were documented in a Certificate of analysis 

provided by the Sponsor and are included in Appendix 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 

This 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 objectives 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 obtained 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

Temperature (°C) Relative 

Humidity (%) Air Change

(per hour)

Mean Light 

Intensity 

Maximum Minimum Maximum Minimum (LUX)

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 feed 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 feed 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 was selected for this study since it is an exaggerated model for

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 15-16
JRF 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 Feed 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 processed 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 were 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 10, 100 and 1000 mg/kg b. wt./day. 

8.3 Clinical Observations (Refer Table 2; Appendix 2) 

All the animals given reverse osmosis water and treated with 10, 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 10, 100 and 1000 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 Feed Consumption (Refer Table 5; Appendix 5) 

No change in feed consumption was observed in rats treated with sodium 3-nitrobenzenesulfonate 

at 10, 100 and 1000 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 Levels Proposed Dose Level-I Proposed Dose Level-II

Low 10 62.5

Mid 100 250

High 1000 1000
JRF 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 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, 3rd edition, Hayes A.W. (Ed), Raven Press Ltd., New York, pp. 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 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 24 of 85 

Repeated Dose 14-Day Oral Toxicity Study of 

 Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

TABLE 1 

Mortality Record 

Group

Dose

(mg/kg 

b. wt./day)

Mortality % Mortality

Male

(N=05)

Female

(N=05)

Total

(N=10) Male Female Total

G1 0 0 0 0 0 0 0

G2 10 0 0 0 0 0 0

G3 100 0 0 0 0 0 0

G4 1000 0 0 0 0 0 0

Key: N = Number of animals 
JRF 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 animals 

 
JRF 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; G3-100; 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; G3-100; 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 sacrifice 

 
JRF 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; G3-100; 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; G3-100; 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 

Feed Consumption (g/rat/day) – Group Mean Values 

Dose: G1-0; G2-10; G3-100; 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

8-11 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; G3-100; 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 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 20.82 1.56 2

Keys: SD = Standard deviation, n = Number of cages, TS = Terminal sacrifice 

 
JRF Study Number: 410-1-04-5132 (Final Report) Page 29 of 85 

Mean Body Weight (g) - Male (G1 to G4)

150.00

160.00

170.00

180.00

190.00

200.00

210.00

220.00

230.00

240.00

250.00

1 4 8 11 TS

Day

Mean Body Weight (g)

G1 G2 G3 G4

Repeated Dose 14-Day Oral Toxicity Study of 

Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

FIGURE 1 

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

Mean Body Weight (g) - Female (G1 to G4)

120.00

130.00

140.00

150.00

160.00

170.00

180.00

1 4 8 11 TS

Day

Mean Body Weight (g)

G1 G2 G3 G4

Repeated Dose 14-Day Oral Toxicity Study of 

Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

FIGURE 2 

Mean Body Weight (g) G1 to G4 – Female 
JRF Study Number: 410-1-04-5132 (Final Report) Page 31 of 85 

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

0.00

10.00

20.00

30.00

40.00

50.00

60.00

(1-4) (1-8) (1-11) (1-TS)

Day

Mean Body Weight Change (%)

G1 G2 G3 G4

Repeated Dose 14-Day Oral Toxicity Study of 

Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

FIGURE 3 

Mean Body Weight Change (%) (G1 to G4) – Male 
JRF Study Number: 410-1-04-5132 (Final Report) Page 32 of 85 

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

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

 (1-4) (1-8) (1-11) (1-TS)

Day

Mean Body Weight Change (%)

G1 G2 G3 G4

Repeated Dose 14-Day Oral Toxicity Study of 

Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

FIGURE 4 

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

Repeated Dose 14-Day Oral Toxicity Study of 

Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

FIGURE 5 

Mean Feed Consumption (g/rat/day) G1 to G4 - Male Mean Feed Consumption (g/rat/day) - Male (G1 to G4)

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

" (1-4)" " (4-8)" " (8-11)" " (11-TS)"

Day

Mean Feed Consumption (g/rat/day)

G1 G2 G3 G4
JRF Study Number: 410-1-04-5132 (Final Report) Page 34 of 85 

Repeated Dose 14-Day Oral Toxicity Study of 

Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

FIGURE 6 

Mean Feed Consumption (g/rat/day) G1 to G4- Female 

 Mean Feed Consumption (g/rat/day) - Female (G1 to G4)

0.00

5.00

10.00

15.00

20.00

25.00

" (1-4)" " (4-8)" " (8-11)" " (11-TS)"

Day

Mean Feed Consumption (g/rat/day)

G1 G2 G3 G4
JRF Study Number: 410-1-04-5132 (Final Report) Page 35 of 85 

Repeated Dose 14-Day Oral Toxicity Study of 

 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 Day

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

11

12

13

14

15

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 sacrifice 
JRF 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

21

22

23

24

25

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

TS

TS

TS

TS

15

15

15

15

15

Group: G4; Dose: 1000 mg/kg b. wt./day

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

31

32

33

34

35

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 sacrifice 
JRF Study Number: 410-1-04-5132 (Final Report) Page 37 of 85 

Repeated Dose 14-Day Oral Toxicity Study of 

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 = Normal 
JRF 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

11 1 1 1 1 1 1 1 1 1 1 1 1 1 1

12 1 1 1 1 1 1 1 1 1 1 1 1 1 1

13 1 1 1 1 1 1 1 1 1 1 1 1 1 1

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1

15 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

16 1 1 1 1 1 1 1 1 1 1 1 1 1 1

17 1 1 1 1 1 1 1 1 1 1 1 1 1 1

18 1 1 1 1 1 1 1 1 1 1 1 1 1 1

19 1 1 1 1 1 1 1 1 1 1 1 1 1 1

20 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Key: 1 = Normal 
JRF 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

21 1 1 1 1 1 1 1 1 1 1 1 1 1 1

22 1 1 1 1 1 1 1 1 1 1 1 1 1 1

23 1 1 1 1 1 1 1 1 1 1 1 1 1 1

24 1 1 1 1 1 1 1 1 1 1 1 1 1 1

25 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

26 1 1 1 1 1 1 1 1 1 1 1 1 1 1

27 1 1 1 1 1 1 1 1 1 1 1 1 1 1

28 1 1 1 1 1 1 1 1 1 1 1 1 1 1

29 1 1 1 1 1 1 1 1 1 1 1 1 1 1

30 1 1 1 1 1 1 1 1 1 1 1 1 1 1

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

APPENDIX 2 (Continued)

Group: G4; Dose: 1000 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

31 1 1 1 1 1 1 1 1 1 1 1 1 1 1

32 1 1 1 1 1 1 1 1 1 1 1 1 1 1

33 1 1 1 1 1 1 1 1 1 1 1 1 1 1

34 1 1 1 1 1 1 1 1 1 1 1 1 1 1

35 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

36 1 1 1 1 1 1 1 1 1 1 1 1 1 1

37 1 1 1 1 1 1 1 1 1 1 1 1 1 1

38 1 1 1 1 1 1 1 1 1 1 1 1 1 1

39 1 1 1 1 1 1 1 1 1 1 1 1 1 1

40 1 1 1 1 1 1 1 1 1 1 1 1 1 1

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

Repeated Dose 14-Day Oral Toxicity Study of 

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 156.3 173.4 198.5 215.0 236.9

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 127.8 132.8 142.7 158.2 166.4
JRF 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: 1000 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 127.4 133.6 142.8 151.7 159.3
JRF Study Number: 410-1-04-5132 (Final Report) Page 43 of 85 

Repeated Dose 14-Day Oral Toxicity Study of 

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 days 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 30.20
JRF 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 25.10 30.42 43.26 30F 7.57 16.33 21.20 29.40

Group: G4; Dose: 1000 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 28.23 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 25.04
JRF 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 

Feed Consumption (g/rat/day) of Individual Rat 

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

Cage N° 

& Sex

During days Cage N° 

& Sex

During days

1 - 4 4 - 8 8 - 11 11 -TS 1 - 4 4 - 8 8 - 11 11-TS

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

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

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

Cage N° 

& Sex

During days Cage N° 

& Sex

During days

1 - 4 4 - 8 8 - 11 11 -TS 1 - 4 4 - 8 8 - 11 11 -TS

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

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

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

Cage N° 

& Sex

During days Cage N° 

& Sex

During days

1 - 4 4 - 8 8 - 11 11 -TS 1 - 4 4 - 8 8 - 11 11 -TS

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

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

Group: G4; Dose: 1000 mg/kg b. wt./day 

Cage N° 

& Sex

During days Cage N° 

& Sex

During days

1 - 4 4 - 8 8 - 11 11 -TS 1 - 4 4 - 8 8 - 11 11 -TS

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

14M 18.68 20.17 22.38 29.50 16F 13.61 14.02 14.98 19.72

JRF 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.Sc., D.M.L.T.

SNEHA VINODAN, M.Sc. M.L.T. 

Statistician : RAJESH PATEL, 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 ...................................................................................... 54
JRF 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 Dose Group (10 mg/kg b.wt./day)

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

G4 - High Dose Group (1000 mg/kg b.wt./day)

kg - Kilogram

mg - Milligram - 

NAD - No Abnormality Detected

N - Number of Animals

N° - Number 

SD - Standard Deviation 
JRF 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, 

statistically 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 

Parameters Absolute organ weight (g) Organ weight relative 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

Mean 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 0.245 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

1000 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 55 5

Internal

No Abnormality Detected 5 5 5 5 5 5 5 5
JRF 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

2 231.8 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 

body 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 0.268
JRF 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 2.848 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: 1000 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

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 0.227
JRF 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 Internal

6 TS NAD NAD

7 TS NAD NAD

8 TS NAD NAD

9 TS NAD NAD

10 TS NAD NAD
JRF 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

11 TS NAD NAD

12 TS NAD NAD

13 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

16 TS NAD NAD

17 TS NAD NAD

18 TS NAD NAD

19 TS NAD NAD

20 TS NAD NAD
JRF 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

External Internal

26 TS NAD NAD

27 TS NAD NAD

28 TS NAD NAD

29 TS NAD NAD

30 TS NAD NAD

 
JRF Study Number: 410-1-04-5132 (Final Report) Page 57 of 85 

APPENDIX 6 (Continued) 

APPENDIX B (Continued) 

Group: G4; Dose: 1000 mg/kg b. wt./day Sex: Male 

Rat N°

Mode

of

Death

Gross Findings

External Internal

31 TS NAD NAD

32 TS NAD NAD

33 TS NAD NAD

34 TS NAD NAD

35 TS NAD NAD

Group: G4; Dose: 1000 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 NAD
JRF 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 Report 
JRF 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 Feed, Water and Paddy Husk 

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

APPENDIX 8 (Continued) 

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

APPENDIX 8 (Continued) 

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

Repeated Dose 14-Day Oral Toxicity Study of 

Sodium 3-nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study) 

APPENDIX 9 

Results of Microbiological Analysis of Rat Pellet Feed, Water and Paddy Husk 

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

APPENDIX 9 (Continued) 

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

APPENDIX 9 (Continued) 

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

APPENDIX 9 (Continued) 

C. Microbial Analysis Report of Bedding Material 
JRF 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 - 2012
JRF 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 ............................................................................................................................. 9 
JRF 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 ............................................................................................................................. 14
JRF 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 Toxicopathologist

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 Schedule

 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% (Information provided by the Sponsor via CoA) 

Batch N° : 50623

Supplied by : Reservol LP
JRF 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 

identification provided by the Sponsor will be responsibility of the Sponsor.

Storage Condition (at JRF) : As per the instruction received from the Sponsor on storage of the test

item, the test item will be stored in its original container as supplied by 

the Sponsor at 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 Deviation 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 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 15-16

7. OBSERVATIONS 

Following observations will be recorded: 

7.1 Clinical 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 conducting 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 identification 
JRF 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° 228-2-13-5312, “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 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 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-nitrobenzensulfonate 
JRF 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 Compliance 
JRF Study Number: 410-1-04-5132 (Final Report) Page 85 of 85 

APPENDIX 12 (Continued)