2012FEB 28 M~ II: 23 

JRF Study Number: 207-2-11-4305 Page 1 of9 

STUDY PLAN 

VAPOUR PRESSURE OF SODIUM 3-NITROBENZENE SULFONATE 

(CAS # 127-68-4, US EPA HPV 3) 

GUIDELINES: OECD N° 104 AND U.S. EPA, OPPTS 830.7950 


TEST: ASTM E 1782-08 (THERMAL ANALYSIS) 


TEST RULE: 40 CFR 799.5089 


SPONSOR 

TIARCO CHEMICAL 


1300 TIARCO DRIVE 


DALTON 


GA30720 


TECHNICAL CONTACT: JEFF VANDERLIP 


REGULATORY CONTACT: BENNETT KING 


PERFORMING LABORATORY 

JAI RESEARCH FOUNDATION 


DEP ARTMENT OF CHEMISTRY 


VALVADA-396108 


DIST. V ALSAD 


GUJARAT 


INDIA 


11111111111 111111111111111111111111111111 11111111111111111111111 

40120000051 

FEBRUARY - 2012 
JRF Study Number: 207-2-11-4305 Page 2 of9 

CONTENTS 


PAGE N° 


TITLE PAGE ........................................................................................................................................... 1 


CONTENTS ............................................................................................................................................. 2 


I. GENERAL INFORMATION ............................................................................................................ 3 


1.1 Study Professional .................................................................................................................... 3 


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


1.3 Technical Contact. .................................................................................................................... 3 


1.4 Regulatory Contact ................................................................................................................... 3 


1.5 Proposed Study Dates ............................................................................................................... 4 


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


2. INTRODUCTION ............................................................................................................................. 4 


2.1 Objective .................................................................................................................................. 4 


2.2 Regulatory Guidelines .............................................................................................................. 4 


2.3 Principle ................................................................................................................................... 4 


2.4 Test Item ................................................................................................................................... 5 


3. GOOD LABORATORY PRACTICE ............................................................................................... 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 .................................................................................................................... 6 


4. EXPERIMENTAL PROCED"URE .................................................................................................... 6 


4.1 Instrument ................................................................................................................................ 6 


4.2 Methodology ............................................................................................................................ 7 


4.2.1 Determination ofVapour Pressure [Effusion Method using TGA] ................................ 7 


4.2.2 Calculation ..................................................................................................................... 7 


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


6. ARCHNES ....................................................................................................................................... 8 


7. REFERENCES .................................................................................................................................. 8 


8. STUDY PLAN APPROV AL ............................................................................................................. 9 

JRF Study Number: 207-2-11-4305 

1. GEl\~RAL INFORMATION 

1.1 Study Professional 

Name of Personnel Qualification Profession Experience 

A.Ho Patel MoSco Chemist 14 years 

(Study Director) 

Ashok S. Amruskar MoSc. Chemist 5 years 

(Dy. Study Director) 

Page 3 of9 

Training 

Declared Study Director for all 

Physico-chemical studies 

Declared Study Director for all 

Physico-chemical studies 

Note: List ofadditional study personnel will be documented in the study file and will be presented in 

the final report. 

1.2 Facility Management 

1.3 Technical Contact 

1.4 Regulatory Contact 

Dr. Manishi Agarwal 

Assistant Director, Department ofChemistry 

Jai Research Foundation, Off. N.H. N°.8 

Near Daman Ganga Bridge 

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

Ph. : + 91-260 654 0242 (Office) 

Ph: +91 9909900929 (Mobile) 

E-mail: agarwalm{@.jrfonline.com 

Jeff Vanderlip 

706/277-1300 

Tiarco Chemical 

1300 Tiarco Drive 

Dalton. Georgia 30720 

Bennett King 

864/457-2321 

Tiarco Chemical 

1300 Tiarco Drive 

Dalton. Georgia 30720 
JRF Study Number: 207-2-11-4305 Page 4 of9 

1.5 Proposed Study Dates 

Experiment Start March 2012* 

Experiment Completion March 2012* 

Draft Report Submission November 01,2012 

Final Report Submission December 10,2012 

Note: (*) = Experiment start and completion date depends on EPA response to the study plan 

1.6 Study Plan and Amendment (if any) Distribution 

a. Original copy in Archive and Study Sponsor; b. Photocopy to Study Director and QAU 

2. INTRODUCTION 

2.1 Objective 

The objective of this study is to determine the vapour pressure of Sodium 3-nitrobenzene sulfonate 

(CAS No. 127-68-4). 

2.2 Regulatory Guidelines 

OECD, 2006: OECD N° 104, "Vapour Pressure". The Organisation for Economic Cooperation and 

Development (OECD) guidelines for the Testing ofChemicals, adopted by the Council on March 23, 

2006. 

Standard Test Method for Determining Vapour Pressure by Thermal Analysis, ASTM E 1782-08, 

approved March 1, 2008. 

U.S. EPA, 1996: OPPTS 830.7950 "Vapour Pressure" (EPA 712-C-96-043). The United States 

Environmental Protection Agency, Product Properties Test Guidelines (August 1996). 

2.3 Principle 

The quantity of the test item leaving a cell per unit time through an aperture of known size is 

determined under vacuum conditions such that return of the test item into the cell is negligible (by 

measuring the weight loss). 
JRF Study Number: 207-2-11-4305 	 Page 5 of9 

2.4 	 Test Item 

The Test Item Data Sheet has been completed by the Sponsor. The representative sample ofthe test 

item will be retained for archive purpose. Any residual test item will be disposed off 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-nitrobenzene sulfonate 

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

C.A. Name Benzenesulfonic acid, 3-nitro, sodium salt (1: 1) 

CAS N° 127-68-4 

Batch N° 50623 

Analysed Purity 96.9% 

Manufacture by Tiarco Chemical 

Supplied by Tiarco Chemical 

Date of Manufacture September 08, 2011 

Date of Expiry September 08, 2013 

Ap~earance Yellow to tan granular 

Storage Condition (at JRF) As per the instruction received from the Sponsor on storage ofthe 

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

supplied by the Sponsor at 40 - 90 OF in Test Item Control Office 

(TICO). The stability of the test item in storage will be the 

responsibility of the Sponsor. 

JRF Test Item Code SNB 444 
JRF Study Number: 207-2-11-4305 	 Page 6 of9 

3. 	 GOOD LABORATORY PRACTICE 

3.1 	 GLP Compliance 

This study will be conducted in compliance with the OECD Principles of Good Laboratory Practice 

(as revised in 1997), ENVIMCICHEM(98)17, N° 1, Environment Directorate, Organisation for 

Economic Cooperation and Development, Paris (1998). 

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 

communicated to the Sponsor as soon as practicable (by telephone, facsimile transmission or e-mail) 

and will be confirmed as soon as possible by way of formal study plan amendment. 

3.4 	 Deviation from the Study Plan 

Any deviation from the study plan will be documented in study file and reported in the study report. 

3.5 	 Quality Assurance 

The Quality Assurance Unit (QAU) of JRF will audit the critical phases of the study, the raw data, 

draft and final reports. The audit reports will be provided to the Study Director and the Management. 

The dates of audits and reporting of findings to the Study Director and the Management will be 

incorporated in the study report. 

4. 	 EXPE~NTALPROCEDURE 

4.1 	 Instrument 

Thermogravimetric analyzer (TGA). 
JRF Study Number: 207-2-11-4305 	 Page 7 of9 

4.2 	 Methodology 

4.2.1 	 Determination of Vapour Pressure [Effusion Method using TGA] 

A quantity of 10-50 mg test item will be added to the balance pan ofthe instrument. The balance will 

be allowed to stand for some time to get stabilized. After the balance is stabilized, the reading will be 

recorded electronically. The temperature scanning will be started between the ranges of40 to 70°C 

and will be scanned for a range of 10°C using nitrogen as purging gas @ 50mLlmin. The mass (mg) 

and the temperature readings (0C) will be recorded automatically into a computer file and the report 

will be printed. The readings oftemperature and mass will be converted into Kelvin (K) and Kilogram 

(kg), respectively. The experiment will be performed in triplicate and the mean value will be reported. 

4.2.2 	 Calculation 

The vapour pressure will be calculated at each set temperature using the following formula: 

Vp A 

Where, 

Vp = Vapour pressure (Pa) 


om Mass difference (kg) 


g Acceleration due to gravity (9.813 mls2

) 


A Area of the orifice (3.14 x 10.6 m2

) 


After calculating the vapour pressure (Pa) the LoglO of the value obtained will be calculated. 

A graph will be plotted ofreciprocal temperature (11K) versus LoglO [Vp(Pa)] to obtain a straight line, 

intercept and slope. 

To obtain the vapour pressure at a particular temperature the following formula will be used: 

Slope LoglO [Vp(Pa)] ----'-----+ Intercept Temperature (K) 

After calculating the LoglO[Vp(Pa)] at the particular temperature, the anti log of the mean of the 

replications will be calculated and reported. 
JRF Study Number: 207·2·11-4305 PageS of9 

5. REPORT 

Unless otherwise requested by the Sponsor, one copy ofthe fmal report will be issued along with one 

soft copy in PDF. The report will include the following information: 


Description of the test item including certificate of analysis 


Details ofthe experimental procedure 


Results 


Personnel involved in the study 


Thermographs 


The signed study plan and study plan amendment (if any) 


Records ofdeviation(s) from the study plan (if any) 


6. ARCIDVES 

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

recorded data, documentation, the signed study plan, any study plan amendment, thermographs, the 

draft report, a copy ofthe final report and the representative sample ofthe test item will be retained in 

the GLP Archives at Jai Research Foundation for a period often years. At the end ofthis 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 off. 

7. REFERENCES 

OECD, 1998: OECD Series on Principles ofGood Laboratory Practice and Compliance Monitoring, 

Number 1, "OECD Principles on Good Laboratory Practice" ENVIMC/CHEM(98)17 (as revised in 

1997). 

OECD, 2006: OECD N° 104, "Vapour Pressure". The Organisation for Economic Cooperation and 

Development (OECD) guidelines for the Testing ofChemicals, adopted by the council on March 23, 

2006. 

Standard Test Method for Determining Vapour Pressure by Thermal Analysis, ASTM E 1782-08, 

approved March 1, 2008. 

U.S. EPA, 1996: OPPTS 830.7950 "Vapour Pressure" (EPA 712-C-96-043). The United States 

Environmental Protection Agency, Product Properties Test Guidelines (August 1996). 

Vohra.Y, 2006: Validation ofEffUsion Method for Determination ofVapour Pressure ofEthofumesate 

Technical, JRF Study Number 5874, Jai Research Foundation, Valvada - 396 108, Gujarat, India. 
JRF Study Number: 207-2-11-4305 	 Page 9 of9 

8. 	 STUDY PLAN APPROVAL 

We. the undersigned have read the whole study plan for "Vapour Pressure of Sodium 

3-Nitrobenzene Sulfonate (CAS # 127-68-4, US EPA HPV 3)" and confml1 that the study will be 

performed as per this study plan. 

Study Director 	 A.I1. PATEL 

~'A~'-' 

Fi t:24J 20 J2­

Facility Management 	 DR. l\lANISIlJ AGARWAL Signature and Date 

For Study Sponsor 	 TlARCO VANDERLIP 

DALTON, GA 30720 

Name of Sponsor's Representative 

Signature Bnd Date 
2012FE828 p.:111:23 

JRF Study Number: 496-1-04-4306 Page lof17 

STUDY PLAN 

REPRODUCTION/ DEVELOPMENTAL TOXICITY SCREENING TEST OF 


SODIUM 3- NITROBENZENE SULFONATE (CAS # 127-68-4; US EPA HPV 3) IN 


WISTAR RATS 


GUIDELINES: OPPTS 870.3550 


TEST: 40 CFR 799.9355 


TEST RULE: 40 CFR 799.5089 


SPONSOR 

TIARCO CHEMICAL 

1300 TIARCO DRIVE 

DAL TON, GA 30720 

TECHNICAL CONTACT JEFF VANDERLIP 

REGULATORY CONTACT BENNETT KING 

PERFORMING LABORATORY 

JAI RESEARCH FOUNDATION 


DEPARTMENT OF TOXICOLOGY 


VALVADA-390 108 


DIST. VALSAD 


GUJARAT 


INDIA 


FEBRUARY - 2012 

JRF Study Number: 496-1-04-4306 Page 2 oft7 

CONTENTS 

PAGE N° 


TITLE PAGE ................................................................................................................................................ 1 


CONTENTS ..................................................................................................................................................2 


1. GENERAL INFORMATION .............................................................................................................4 


1.1 Study Personnel ........................................................................................................................4 


1.2 Facility Management : ..............................................................................................................4 


1.3 Technical Contact : ...................................................................................................................4 


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


1.5 Proposed Dates .........................................................................................................................5 


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


3. GOOD LABORATORY PRACTICE (GLP) ...................................................................................... 7 


3.1 GLP Compliance ......................................................................................................................7 


3.2 Standard Operating Procedures (SOP) .....................................................................................7 


3.3 Amendment to Study Plan ........................................................................................................7 


3.4 Deviation from the Study Plan ................................................................................................. 7 


3.5 Quality Assurance .................................................................................................................... 7 


4. ANIMAL WELFARE ......................................................................................................................... 7 


4.1 Humane Endpoint .....................................................................................................................8 


5. TEST SySTEM...................................................................................................................................8 


5.1 Test System Specification ........................................................................................................ 8 


5.2 Justification of Selection of Test System .................................................................................8 


5.3 Location of the Study ...............................................................................................................8 


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


5.5 Acclimatization ........................................................................................................................9 


5.6 Environmental Conditions ........................................................................................................9 


5.7 Housing ....................................................................................................................................9 


5.8 Diet and Water ..........................................................................................................................9 


5.9 Grouping...................................................................................................................................9 


5.10 Animal Identification ............................................................................................................. 10 


5.11 Mating Procedure ................................................................................................................... 1 0 

JRF Study Number: 496·1-04-4306 Page 3 of 17 


CONTENTS (Continued) 

PAGE N° 

6. EXPER.Th1ENTAL PROCEDURE.................................................................................................... 1 0 


6.1 Vehicle and Justification for Choice ...................................................................................... 10 


6.2 Route and Mode of Administration and Justification for Choice ........................................... 1 0 


6.3 Dose Levels and Justification ................................................................................................. 10 


6.4 Preparation of Dose Formulations .......................................................................................... 11 


6.5 Stability of the Test Item in the Vehicle ................................................................................. 11 


6.6 Homogeneity and Active Ingredient Concentration of the Test Item in the Vehicle ............. 11 


6.7 Administration of Doses......................................................................................................... 11 


6.8 Experimental Schedule ........................................................................................................... 11 


6.9 Summary of the Experimental Design, Group and Number of Animals................................ 11 


7. OBSERVATIONS ............................................................................................................................. 12 


7.1 Parental Rat ............................................................................................................................ 12 


7.1.1 Mortality and Morbidity ............................................................................................. 12 


7.1.2 Clinical Signs .............................................................................................................. 12 


7.1.3 Body Weights ............................................................................................................. 12 


7.1.4 Food Consumption ..................................................................................................... 12 


7.2 Pups ........................................................................................................................................ 12 


7.2.1 Pup Body Weight. ....................................................................................................... 12 


7.3 Necropsy................................................................................................................................. 13 


7.4 Organ Weight ......................................................................................................................... 13 


7.5 Preservation of OrganslTissues .............................................................................................. 13 


7.6 Histopathology ....................................................................................................................... 13 


8. DATA RECORDING AND EVALUATION ................................................................................... 14 


8.1 Data Recording ....................................................................................................................... 14 


8.2 Data Evaluation ...................................................................................................................... 14 


9. REPORT............................................................................................................................................ 14 


10. ARCHIVES ....................................................................................................................................... 15 


11. REFERENCES .................................................................................................................................. 16 


12. STUDY PLAN APPROVAL ............................................................................................................ 17 

JRF Study Number: 496-1-04-4306 	 Page 4of17 

1. 	 GENERAL INFORMATION 

1.1 	 Study Professionals 

Name of Personnel Qualification Profession Experience Training 

Deepak Ujawane M. Ph anna. Toxicologist 3 years Declared Study Director for all 

(Study Director) Development and reproductive 

toxicity studies 

Manish V. Patel Ph.D. Toxicologist 14 years 	 Declared Study Director for all 

Development and reproductive 

toxicity studies 

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

for animals handling 

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

Sanjay Divedia M.Sc. Chemist 10 years 	 Trained for dose fonnulation 

analysis 

Noe: 	 List of additional study personnel will be documented in the study file and will be 

presented in the final report. 

1.2 	 Facility Management T. G. Marvania, Ph.D. 

Assistant Director, Department of Toxicology, 

Jai Research Foundation, Off. N.H. N°.8, 

Near Daman Ganga Bridge, 

Valvada. 396 108, Dist. Val sad, Gujarat, India. 

Ph. : + 91-260 654 0242 (Office), 

Ph: +91 99099 00913 (Mobile), 

E-mail: marvaniatg@jrfonline.com 

1.3 	 Technical Contact Jeff Vanderlip 

706/277-1300 

Tiarco Chemical 

1300 Tiarco Drive 

Dalton, Georgia 30720 

1.4 	 Regulatory Contact Bennett King 

864/457-2321 

Tiarco Chemical 

l300 Tiarco Drive 

Dalton, Georgia 30720 
JRF Study Number: 496-1·04·4306 	 Page 5 of17 

1.5 	 Proposed Dates 

Experiment Start March 2012 (*) 

Treatment Start Will be filled after confirmation of start date 

Mating Start Will be filled after confirmation of start date 

Terminal Sacrifice Start Will be filled after confirmation of start date 

Experiment Completion Will be filled after confirmation of start date 

Interim Progress Submission Will be filled after confirmation of start date 

Draft Report Submission November 1,2012 

Final Report Submission December 10, 2012 

Note: (*) Experiment start date depends on EPA response to the study plan 

1.6 	 Study Plan and Amendment (if any) Distribution 

a. Original copy in Archive and Study Sponsor; 

b. Photocopy to Study Director, QAU and Pathology 

2. 	 INTRODUCTION 

2.1 	 Objective 

The objective of this Screening Test is to estimate the initial information on possible effects ofthe 

sodium 3- nitrobenzene sulfonate (CAS No. 127-68-4) on reproduction and/or development, 

either at an early stage of assessing the toxicological properties of chemicals or on chemicals of 

concern. 

2.2 	 Regulatory Guidelines 

This Study will be conducted in compliance with the guidelines of: 

The United States Environmental Protection Agency (1998). Health Effects Test Guidelines 

OPPTS 	 870.3550, EPA712-C-00-367, July 2000. "ReproductionlDevelopmental Toxicity 

Screening Test". 

2.3 	 Test Method Principle 

The sodium 3 -nitrobenzene sulfonate (CAS No. 127-68-4) will be administered in graduated 

doses to three groups of males and females. Males will be dosed for a minimum of four weeks 

(this includes a minimum of two weeks prior to mating, during the mating period and, 

approximately, two weeks post-mating) and up to and including the day before scheduled 

sacrifice (One day after terminal sacrifice of all the females). Females will be dosed throughout 

the study (this includes two weeks prior to mating, the variable time to conception, the duration of 

pregnancy and at least four days after delivery up to and including the day before scheduled 

sacrifice (post-partum 3). 
JRF Study Number: 496-1-04-4306 	 Page 6 oft7 

During the period of test item administration, the animals will be observed closely each day for 

signs of toxicity. Animals, which die or will be sacrificed under moribund condition during the 

test period, are necropsies and at the time of terminal sacrifice all the surviving animals will be 

sacrificed. 

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 supplied by the study Sponsor) are as follows: 

Test Item Name Sodium 3- nitrobenzene sulfonate 

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

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

CAS N° 127-68-4 

Batch N° 50623 

Concentration I Purity 96.9% 

Manufactured by Tiarco Chemical 

Supplied by Tiarco Chemical 

Date of Manufacture September 08, 2011 

Date of Expiry September 08, 2013 

Appearance Yellow to tan granular 

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

the test item, the test item will be stored in its original 

container as supplied by the Sponsor at 40 - 90 OF in the Test 

Item Control Office (TICO). The stability of test item in 

storage will be the responsibility of the Sponsor. 

JRF Test Item Code 	 SNB444 
JRF Study Number: 496-1-04-4306 Page 7 of 17 

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), ENVIMC/CHEM (98)17, N° I, Environment Directorate, 

Organization for Economic Co-operation and Development, Paris (1998). 

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. 

3.5 Quality Assurance 

The Quality Assurance Unit (QAU) of JRF will audit the critical phases of the study, the raw 

data, draft and final reports. The audit reports will be provided to the Study Director and the 

Management. The dates of audits and reporting of findings to the Study Director and the 

Management will be incorporated in the study report. 

4. ANIMAL WELFARE 

The study will be 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 will enhance animal well being and experimental quality for the 

advancement of biological knowledge that is relevant to humans and animals. 
JRF Study Number: 496-1-04-4306 	 Page 8 of 17 

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 Foundations policy on animal 

welfare. 

Project proposal for the experimentation is subject to approval by Institutional Animal Ethics 

Committee (IAEC), Jai Research Foundation. 

4.1 	 Humaue Endpoint 

Moribund animals or animals obviously in pain or showing signs of severe and enduring distress 

shall be humanely killed, and are considered in the interpretation of the test results in the same 

way as animals that died on test. 

5. 	 TEST SYSTEM 

5.1 	 Test System Specification 

Healthy young adult rats (Rattus norvegicus) of Wistar strain (RccHan:WIST) will be obtained 

from the Animal Breeding Facility, Jai Research Foundation and used for the experiment. Female 

rats wi11 be nulliparous and non-pregnant. At the initiation of the acclimatization rats will of 10 to 

I I weeks old. Care will be taken to avoid mating of siblings in the P generation. 

After acclimatization, healthy 65 male and 65 female rats will be selected and randomized into 

the test groups. Animals not selected for the study will be returned to the animal breeding facility, 

JRF or discarded without any further examination. 

5.2 	 Justification of Selection of Test System 

The rat is the preferred test system because it is a readily available laboratory model and is 

historically shown as acceptable model for developmental and reproductive toxicity testing and 

recommended by the OPPTS and other regulatory authorities. 

5.3 	 Location of the Study 

During the study period, the test animals will be located in the barrier maintained rodent facility 

(BMR) and the experimental room number will be specified in the study schedule. 

5.4 	 Experimental Room Sanitation 

Each day, floor of experimental room will be swept and all work tops and floor will be cleaned 

with a disinfectant solution. Detail of disinfectant will be document in the raw data and reported 

in the final report. 
JRF Study Number: 496-1-04-4306 	 Page 9 of 17 

5.5 	 Acclimatization 

The animals will be received into the experimental room and acclimatized for a minimum period 

of 5 days prior to the cohabitation. During the acclimatization period, the animals will be 

observed daily for clinical signs. Prior to cohabitation, Animals with any evidence of ill health or 

poor physical condition will be excluded from the study. 

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 hours light and 12 hours 

darkness, light hours being 06.00 - 18.00 hours and air changes will be minimum of 15 per hour. 

5.7 	 Housing 

Throughout experiment period, the rats will be housed individually. Except during the mating 

period, the rats will be housed in groups of2 rats/cage (one male plus one female). Mated female 

rat will be caged individually and nesting material (shredded paper) at near term or around 14th 

day of gestation will be provided. During study, the rats will be housed in solid floor 

polypropylene rat cages (size: 41cm 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. The bottom of the cages will be layered with clean sterilized rice 

(paddy) husk as the bedding. 

Cages will be placed on 5/6 tier racks. Cages will be changed at least twicel thrice a week. 

Contaminant analysis of samples of the bedding material will be performed at 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 ad libitum with standard rodent pellet diet with an unlimited 

supply of clean and filtered drinking water (Reverse Osmosis water filter system) in 

polypropylene bottles. 

Every food consignment received is accompanied with a certificate of analysis of nutrient content 

from the food supplier. Quality of food and water is being monitored regularly at Jai Research 

Foundation. The most recent result of microbial and chemical contaminant analysis will be 

reported in the final report. 

5.9 	 Grouping 

The rats will be randomly assigned to four groups using a stratified body weight procedure (SOP 

N° JRFISSVSOP - 829) sex-wise. Prior to the start of the treatment, the body weight of the rat 

will not exceed ± 20% of the mean body weight in each sex. Rats out of this weight range will be 

returned to the animal breeding facility, JRF or discarded without any further examination. 
JRF Study Number: 496-1-04-4306 	 Page 10 of17 

5.10 	 Animal Identification 

During acclimatization, a temporary animal number will be marked with indelible non toxic 

marker pen on the dorsal surface of the tail. After grouping, each rat will have permanent animal 

number and marking system. Individual rat will be identified with unique numbers tattooed on the 

tail of each animal using tattoo machine, as per SOP N° JRF/TOXISOP-2041 and 2042. Each 

animal will be individually housed in cage on confirmation of mating. Each cage will be labeled 

with a card showing: 

Study N°, Study code, Test Item code, Group N° and Sex, Dose (mg/kg b. wt.lday), Animal N°. 

5.11 	 Mating Procedure 

During the mating period, the male and female rats will be mated in a I: I mating ratio to procure 

the F I generation. Each morning, the females will be examined for the presence of sperm and the 

'day 0' of pregnancy will be recorded. 'Day 0' of pregnancy is defined as the day on which 

sperms are observed in the vaginal smears of rats. After evidence of copulation, mated female rats 

will be caged individually. 

For each mating, each female will be placed with a single male from the same dose level 

(I: 1 mating) until copUlation occurs or 2 weeks have elapsed. If mating has not occurred during 

two weeks mating period, re-mating of females with proven males of the same group may be 

performed. Detail record of the mating pairs will be maintained in the raw data. Mating of 

siblings will be avoided. 

6. 	 EXPERIMENTAL PROCEDURE 

6.1 	 Vehicle and Justification for Choice 

The vehicle will be selected based on data of the solubility check performed at JRF and after 

consultation with the Sponsor. 

6.2 	 Route and Mode of Admiuistratiou aud Justification for Choice 

The route of Sodium 3- nitrobenzene sulfonate (CAS No. 127-68-4) administration will be 

through oral gavage since it will provide an exaggerated model of the reasonably foreseeable 

exposure in Human. 

6.3 	 Dose Levels and Justification 

The dose levels for main study will be selected based on the dose range finding study result of the 

test item and will be intimated to the Sponsor through a formal study plan amendment. 
JRF Study Number: 496-1-04-4306 	 Page 11 of17 

6.4 	 Preparation of Dose Formulations 

Required quantity of test item for each dose group will be received from TICO. Test item will be 

formulated in suitable vehicle. The dose formulations (mixture of test item and vehicle) will be 

prepared every day extemporaneously prior to dosing. If require, it will be thoroughly mixed 

using magnetic stirrer or glass rod in order to administer a homogeneous preparation. 

6.5 	 Stability of the Test Item in the Vehicle 

The stability of active ingredient (a.i.) in vehicle will be determined prior to initiation ofthe study 

by using validated analytical method. 

6.6 	 Homogeneity and Active Ingredient Concentration of the Test Item in the Vehicle 

The active ingredient (a.i.) concentration and homogeneity of the Sodium 3- nitrobenzene 

sulfonate (CAS No. 127-68-4) will be analyzed before start and once during the treatment period 

from the main study. Details will be reported in the final report. 

6.7 	 Administration of Doses 

The rats will dosed with the Sodium 3- nitrobenzene sulfonate (CAS No. 127-68-4) daily for 

seven days a week. The dose volume for the administration will be between 5 to 10 mLlkg body 

weight. The volume of dose solution administered will be adjusted at least weekly to maintain a 

constant dose level in the term of animal recent body weight. Dose solution will be prepared 

freshly every day and administered at approximately the same time each day. 

The control group will be a vehicle-control group. The control group will be received the vehicle 

in the highest volume used. 

6.8 	 Experimenta1 Schedule 

Dosing of both the sexes will be initiated at least 2 weeks prior to mating and continued during 

the mating period. After mating, the male rats will be further dosed up to and including the day 

before scheduled sacrifice (One day after terminal sacrifice of all the females). Females will be 

further dosed during pregnancy and up to post-partum day 3. 

6.9 	 Summary of the Experimental Design, Group and Number of Animals 

Group Dose Total N° of Rat Minimum N° of Pregnant Female 

N° (mg/kg b. wt./day) Male Female Rat Require for Study

GI Control (0) 15 15 10 

G2 Low Dose 15 15 10 

G3 Mid Dose 15 15 10 

G4 High Dose 15 15 10 
JRF Study Number: 496-1-04-4306 	 Page 12 of17 

7. 	 OBSERVATIONS 

7.1 	 Parental Rat 

7.1.1 	 Mortality and Morbidity 

Rats will be observed at least twice a day for mortality and morbidity. Rat and pup, which die / 

sacrificed during moribund condition during study, will be weighed and subjected to post-mortem 

examination. Females, which show marked signs of reaction to treatment or premature delivery, 

will be sacrificed and examined as per the tenninally sacrificed animals. 

7.1.2 	 Clinical Signs 

All visible clinical signs, such as changes in skin, fur, eye, mucous membranes as well as 

behavior pattern and abortion, if any will be noted and recorded twice a day, every day 

throughout the period of the experiment. 

7.1.3 	 Body Weights 

lndividual body weight of male and female rats will be recorded on the first day of dosing and at 

least weekly thereafter. During gestation period, females will be weighed at a minimum on 

gestation days 0, 7, 14, and 20 and within 24 hours of parturition (day 0 or 1 post-partum), and 

post-partum 4. Parturition day 0 is defined as the day on which the female littered. At the time of 

sacrifice body weight of all the rats will be recorded. 

7.1.4 	 Food Consumption 

During the pre-mating, gestation and lactation periods food consumption will be measured 

weekly at a minimum. The feed consumption will be detennined by weighing of feed input and 

feed left over. Feed spillage/waste may not be estimated during the period, however evidence of 

excessive spillage should be recorded in the raw data, additional feed will be provided as and 

when required. 

7.2 	 Pups 

Each litter will be examined as soon as possible after delivery to establish the number of pups, 

sex of pups, stillbirths, dead pups and the presence of gross anomalies. Pups found dead if not 

macerated on the day of littering will be examined for possible defects and cause of death and 

preserved. 

7.2.1 	 Pup Body Weight 

lndividual pup body weight will be recorded on lactation days 0 and 4. Individual pups 

identification will be perfonned using indelible ink. 
JRF Study Number: 496-1-04-4306 	 Page 13 of17 

7.3 	 Necropsy 

7.3.1 	 All the survived rat rats will be sacrificed by using over dose of CO2 when they are not longer for 

assessment of reproductive effects. Male rats will be scarified on one day after terminal sacrifice 

of all the females. Female rat along with the pups will be sacrificed on post-partum 4. 

7.3.2 	 At the time of sacrifice or death during the study, all rats and pups will be examined 

macroscopically for any structural abnormalities or pathological changes. Special attention will 

be paid to the organs of the reproductive system. 

7.3.3 	 During sacrifice, number of corpora lutea and implantation sites will be recorded. 

7.4 	 Organ Weigbt 

At the time of sacrifice, the testes and epididymides of all male adult rats will weighed. 

7.5 	 Preservation of Organsrrissues 

At the time of sacrifice, following organs/ tissues of rats will be fixed and stored in a suitable 

medium; 

• 	 Vagina, uterus with oviducts, cervix, seminal vesicles, prostate and coagulating gland 

(preserved in 10% Neutral Buffered formalin) 

• 	 Ovaries, testis and epididymis (preserved in Bouin's fixative or Modified Davison's) 

• 	 Organs with gross lesion (preserved in 10% Neutral Buffered formalin) 

7.6 	 Histopathology 

Detailed histological examination will be performed on the ovaries, testes and epididymides of 

the rats from the highest dose group and the control group. Examinations will be extended to the 

rats of other dosage groups when changes are seen in the highest dose group. 

Detailed testicular histopathological examination (e.g., using Bouin's fixative or Modified 

Davison's, paraffin embedding and transverse sections of 4-5±m thickness) will be conducted 

with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell 

structure. The evaluation will be identified treatment-related effects such as retained spermatids, 

missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells 

into the lumen. Examination of the intact epididymis will be included the caput, corpus, and 

cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis will be 

evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and 

phagocytosis of sperm. Histopathological examination of the ovary should detect qualitative 

depletion of the primordial follicle population. PAS and hematoxylin staining will be used for 

examination of the male reproductive organs. 
JRF Study Number: 496-1-04-4306 	 Page 14 of 17 

8. 	 DA TA RECORDING AND EVALUATION 

8.1 	 Data Recording 

All study observations will be recorded in standard fonnats. Individual animal data will be 

provided. Additionally, all data will be summarized in tabular fonn. 

The findings of this toxicity study will be evaluated in tenns of the observed effects, macroscopic 

and microscopic findings. The evaluation will be included the relationship between the dose of 

the test item and the presence or absence of gross lesions in the identified target organs, 

infertility, clinical abnonnalities, affected reproductive and litter perfonnance, body weight 

changes, effects on mortality and any other toxic effects. 

8.2 	 Data Evaluation 

The findings of this toxicity study will be evaluated in tenns of the observed effects, macroscopic 

and microscopic findings. The evaluation will be included the relationship between the dose of 

the test item and the presence or absence of gross lesions in the identified target organs, 

infertility, clinical abnonnalities, affected reproductive and litter perfonnance, body weight 

changes, effects on mortality and any other toxic effects. 

The test parameters will be analyzed using appropriate statistical techniques using Bartlett's test, 

ANOVA and Dunnett's 't' test, Student's t-test, Chi-square test and Test of significance for 

difference of proportions. Significance between control and treatment groups will be calculated 

with statistical significance of 95% confidence level. 

9. 	 REPORT 

Unless otherwise instructed by the Sponsor, one copy of the final report along with one soft copy 

in PDF will be issued to the Sponsor. The report will include the following infonnation: 

1. Test Item: 

a. Physical nature and, where relevant, physicochemical properties. 

b. Identification data. 

2. Vehicle (if appropriate): Justification for choice of vehicle if other than water. 

3. Test animals: 

a. Species/strain used. 

b. Number, age and sex of animals. 

c. Source, housing conditions, diet, etc. 

d. Individual weights of animals at the start of the test. 
JRF Study Number: 496-1-04-4306 	 Page 15 of17 

4. 	 Test conditions: 

a. 	 Rationale for dose level selection. 

b. 	 Details of test item formulation, achieved concentrations, stability and homogeneity 

ofthe preparation. 

c. 	 Details of the administration of the test item. 

d. 	 Details offood and water quality. 

5. 	 Results (toxic response data by sex and dose): 

a. 	 Time of death during the study or whether animals survived to termination. 

b. 	 Nature, severity and duration of clinical observations (whether reversible or not). 

c. 	 Body weightlbody weight change data. 

d. 	 Food consumption, if applicable. 

e. 	 Effects on reproduction, including information on mating/precoital interval, fertility, 

fecundity and gestation duration. 

f. 	 Effects on offspring, including number of pups born (live and dead), sex ratio, 

postnatal growth (pup weights) and survival (litter size), gross abnormalities and 

clinical observations during lactation. 

g. 	 Body weight at termination and organ weight data for the parental animals. 

h. 	 Necropsy data, including number of implantations and number of corpora lutea. 

i. 	 Calculations of pre- and post implantation loss. 

J. 	 Detailed description of histopathological findings. 

k. 	 Statistical treatment of results, where appropriate. 

6. 	 Discussion of results. 

7. 	 Conclusions 

8. 	 Signed study plan and study plan amendment(s) (if any) 

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

10. ARCHIVES 

On completion ofthe study, all original raw data including any storage medium for electronically 

recorded data, documentation, the signed study plan, any study plan amendment, 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 often years. Tissues, slides and blocks will 

be archived for a period of five 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 off. 
JRF Study Number: 496-1-04-4306 Page 16 of 17 

11. REFERENCES 

40 CFR 799.5089 Chemical Testing Requirements for Third Group of High Production Volume 

Chemicals (HPV 3). 

40 CFR 799.9355 TSCA ReproductionJDevelopmental Toxicity Screening Test. 

Fisher, R.A. and Yates, F. (1963): Statistical Tables for Biological, Agricultural and Medical 

Research. 6th Edition, Oliver and Boyd, London, U.K., p. 146. 

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, 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). 

The United States Environmental Protection Agency (1998). Health Effects Test Guidelines 

OPPTS 870.3550, EPA712-C-OO-367, July 2000. "ReproductionJDevelopmental Toxicity 

Screening Test". 
JRF Stud)' Number: 496-1-04-4306 Page 17 of 17 

12. STUDY PLAN APPROVAL 

We. the undersigned have read the whole study plan for JRF Study "Reproduction! 

Denlopmental Toxicity Screening Test of Sodium 3-Nitrobenzene Sulfonate (CAS # 127-68­

4; US EPA liP\' 3) in Wistar Rats" and confinn that the stud) ,.. ill be performed as per this 

study plan. 

Study Director DEEPAK UJAWANE 

Facility Management DR. T. G. MAR\,ANIA 

For Study Sponsor TIARCO V Al"lDERLlP 

DALTON, GA 30720 

Name of Sponsor's Representath:e 

Signature and Date 
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