## 2012FEB 28**M~** 

## II: 23

JRF StudyNumber: 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: ASTME 1782-08 (THERMAL ANALYSIS) TESTRULE: 40 CFR 799.5089


## SPONSOR TIARCO CHEMICAL 1300 TIARCO DRIVE DALTON GA30720 TECHNICALCONTACT: JEFFVANDERLIP REGULATORY CONTACT: BENNETT KING


## PERFORMINGLABORATORY JAI RESEARCHFOUNDATION DEPARTMENT OFCHEMISTRY VALVADA-396108 DIST. V ALSAD GUJARAT INDIA 11111111111 111111111111111111111111111111 11111111111111111111111 40120000051 

### FEBRUARY

- 2012

### JRF StudyNumber: 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 FacilityManagement
................................................................................................................3 1.3 Technical Contact.
....................................................................................................................3 1.4 Regulatory Contact
...................................................................................................................3 1.5 Proposed Study Dates
...............................................................................................................4 1.6 StudyPlan and Amendment (ifany) 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 QualityAssurance
....................................................................................................................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
.............................................................................................................9JRF 

### StudyNumber: 207-2-11-4305 1.**GEl\~RAL** 

### INFORMATION 1.1 StudyProfessional 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) 

### Page3 of9

Training Declared Study Director for all Physico-chemical studies Declared Study Director for all Physico-chemical studies 

### Note: Listofadditional studypersonnelwill bedocumented 

### in the studyfile andwill 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 JeffVanderlip 706/277-1300 Tiarco Chemical 1300 Tiarco Drive Dalton. Georgia 30720 BennettKing 864/457-2321 Tiarco Chemical 1300 Tiarco Drive Dalton. Georgia 30720JRF 

### 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 (ifany) Distribution
a. Original copy in Archive and Study Sponsor; b. Photocopy to Study Director and QAU 

### 2. INTRODUCTION 2.1 Objective The objective ofthis study is to determine the vapourpressure ofSodium 3-nitrobenzene sulfonate (CAS No. 127-68-4). 

### 2.2 Regulatory Guidelines OECD, 2006: OECDN° 104, "VapourPressure". The Organisation for Economic Cooperation and Development(OECD) guidelines fortheTestingofChemicals, adopted bytheCouncilonMarch 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 ofthe 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 offat JRF afterthe 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 ofManufacture September 08, 2011 Date ofExpiry September 08, 2013**Ap~earance** Yellowto tan granular Storage Condition (at JRF) As perthe 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 ofthe 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 compliancewith the OECD Principles ofGood LaboratoryPractice (as revised in 1997), ENVIMCICHEM(98)17, N° 1, Environment Directorate, Organisation for Economic Cooperation and Development, Paris (1998). 3.2 Standard OperatingProcedures (SOP) Unless otherwise specifiedall proceduresmentionedin the studyplan are subjectto detailed Standard Operating Procedures ofJai Research Foundation. 3.3 Amendmentto StudyPlan This study plan may be subject to amendment. Amendment to study plan, whether initiated bythe Sponsororthe Study Directorwillbegenerated, authorizedbythe StudyDirectorand willbe sentto the Sponsorfor approval. In the event that circumstances dictate immediate action, the nature ofthese circumstances will communicatedto the Sponsoras soon as practicable (bytelephone, facsimile transmission ore-mail) and will be confirmed as soon as possible by way offormal study plan amendment. 3.4 Deviation from the StudyPlan Any deviation from the studyplan will be documented in studyfile and reported 

### in the studyreport. 3.5 QualityAssurance The Quality Assurance Unit (QAU) ofJRF will audit the critical phases ofthe study, the raw data, draftand final reports. Theauditreports will beprovidedtothe Study DirectorandtheManagement. The dates ofaudits and reporting offindings 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 Page7 of9 4.2 Methodology 4.2.1 Determination ofVapourPressure [Effusion Method using TGA] Aquantity of 10-50 mg test item will be added to the balance pan ofthe instrument. The balance will be allowedto stand for sometime to get stabilized. Afterthe 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 arange of 10°Cusing nitrogen as purging gas @50mLlmin. The mass (mg) and the temperature readings (0C) 

### will be recorded automatically into acomputer file and the report will beprinted. Thereadings oftemperature andmass willbeconvertedinto Kelvin (K) 

### andKilogram (kg), respectively. Theexperimentwill beperformedintriplicate andthe meanvalue willbereported. 4.2.2 Calculation The vapour pressure will be calculated at each settemperature 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 ofthe orifice (3.14 x 10.6 

### m 2) 

### After calculating the vapourpressure (Pa) the LoglO 

### ofthe value obtained will be calculated. Agraphwill

### beplotted ofreciprocal temperature (11K) versus LoglO 

### [Vp(Pa)] to obtain astraightline, intercept and slope. To obtain the vapourpressure at a particulartemperature 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 ofthe mean ofthe replications will be calculated and reported.JRF Study Number: 207·2·11-4305 PageS of9 5. REPORT Unlessotherwise requested bythe Sponsor, onecopyofthe fmal reportwill be issued alongwith one soft copy in PDF. The report will include the following information: Description ofthe test item including certificate ofanalysis Details ofthe experimental procedure Results Personnel involved in the study Thermographs The signed study plan and study plan amendment (ifany) Records ofdeviation(s) from the study plan (ifany) 6. ARCIDVES 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, thermographs, the draftreport, a copyofthe final reportand the representative sampleofthetestitem will be retained in the GLP Archives at Jai Research Foundation for a period oftenyears. Atthe endofthis 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: OECDSerieson PrinciplesofGood LaboratoryPracticeand ComplianceMonitoring, Number 1, "OECDPrinciples on Good Laboratory Practice"ENVIMC/CHEM(98)17 (asrevised in 1997). OECD, 2006: OECDN° 104, "VapourPressure". The Organisation for Economic Cooperation and Development(OECD)guidelinesforthe TestingofChemicals, adoptedbythecouncil 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: ValidationofEffUsion MethodforDetermination of VapourPressureofEthofumesate 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 studywill be performed as per this study plan.

Study Director 

### A.I1. PATEL

### ~'A~'-' 

## Fi

# t:24J20

## J 2­ Facility Management DR. l\lANISIlJ AGARWAL Signature and Date

For Study Sponsor TlARCO VANDERLIP DALTON, GA 30720

Name ofSponsor's Representative

Signature Bnd Date

## 2012FE828 p.:111:23

JRF 

### Study Number: 496-1-04-4306 Page 

### lof17 

## STUDY PLAN REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENINGTEST OF SODIUM 3- NITROBENZENE SULFONATE (CAS # 

### 127-68-4; US EPA HPV 3) IN WISTARRATS
GUIDELINES: OPPTS 870.3550 TEST: 40 CFR799.9355 TEST RULE: 40 CFR 799.5089


## SPONSOR TIARCO CHEMICAL 1300 TIARCO DRIVE DALTON, GA 30720 TECHNICAL CONTACT JEFF VANDERLIP REGULATORYCONTACT BENNETT KING


## PERFORMING LABORATORY JAIRESEARCH FOUNDATION DEPARTMENT OFTOXICOLOGY VALVADA-390 108 DIST. VALSAD GUJARAT INDIA
FEBRUARY

- 2012

### JRF Study Number: 496-1-04-4306 

### Page2 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 (ifany) 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 ofSelection ofTest System
................................................................................. 8 5.3 Location ofthe Study
...............................................................................................................8 5.4 Experimental Room Sanitation
.................................................................................................8 5.5 Acclimatization
........................................................................................................................9 5.6 Environmental Conditions
........................................................................................................9 5.7 Housing
....................................................................................................................................9 5.8 Dietand Water
..........................................................................................................................9 5.9 Grouping
...................................................................................................................................9 5.10 Animal Identification
............................................................................................................. 10 5.11 Mating Procedure
................................................................................................................... 1 0JRF Study Number: 496·1-04-4306 Page 3 of17 CONTENTS (Continued) 

### PAGEN° 6. EXPER.Th1ENTAL PROCEDURE
.................................................................................................... 1 0 6.1 Vehicle and Justification for Choice
...................................................................................... 10 6.2 Route and Mode ofAdministration and Justification for Choice
........................................... 1 0 6.3 Dose Levels and Justification
................................................................................................. 10 6.4 Preparation ofDose Formulations
.......................................................................................... 11 6.5 Stability ofthe Test Item in the Vehicle
................................................................................. 11 6.6 Homogeneity and Active Ingredient Concentration ofthe Test Item in the Vehicle
............. 11 6.7 Administration ofDoses
......................................................................................................... 11 6.8 Experimental Schedule
........................................................................................................... 11 6.9 Summary ofthe Experimental Design, Group and Number ofAnimals
................................ 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 BodyWeight.
....................................................................................................... 12 7.3 Necropsy
................................................................................................................................. 13 7.4 Organ Weight
......................................................................................................................... 13 7.5 Preservation ofOrganslTissues
.............................................................................................. 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 1 1. REFERENCES
.................................................................................................................................. 16 12. STUDY PLAN APPROVAL
............................................................................................................ 17

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

### 1. GENERAL INFORMATION 1.1 StudyProfessionals

Name ofPersonnel Qualification Profession Experience Training Deepak Ujawane M. Phanna. Toxicologist 3 years Declared Study Director for all 

### (StudyDirector) Development and reproductive toxicity studies Manish V. Patel Ph.D. Toxicologist 14 years Declared Study Directorfor all Development and reproductive toxicity studies Mukesh P. Posia M.V.Sc. Veterinarian 2 years Trained as study veterinarian for animals handling ArabindaAdak M.V.Sc. Pathologist 5years 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 FacilityManagement T. G. Marvania, Ph.D. Assistant Director, Department ofToxicology, Jai Research Foundation, Off. N.H. N°.8, Near Daman Ganga Bridge, Valvada. 396 108, Dist. Valsad, Gujarat, India. Ph. : + 91-260 654 0242 (Office), Ph: +91 99099 00913 (Mobile), E-mail: marvaniatg@jrfonline.com 

### 1.3 Technical Contact JeffVanderlip 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 30720JRF 

### StudyNumber: 496-1·04·4306 Page 5 of17 1.5 Proposed Dates Experiment Start March 2012 (*) Treatment Start Will be filled after confirmation ofstart date Mating Start Will be filled after confirmation ofstart date Terminal Sacrifice Start Will be filled after confirmation ofstart date Experiment Completion Will be filled after confirmation ofstart date Interim Progress Submission Will be filled after confirmation ofstart 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 (ifany) Distribution a. Original copy in Archive and Study Sponsor; b. Photocopyto Study Director, QAU and Pathology 

### 2. INTRODUCTION 2.1 Objective The objective ofthis 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 ofassessing the toxicological properties ofchemicals 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 TestMethod 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 offour 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 ofall the females). Females will be dosed throughout the study (this includes two weeks priorto 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 

### StudyNumber: 496-1-04-4306 Page 6 oft7 During the period oftest item administration, the animals will be observed closely each day for signs oftoxicity. Animals, which die or will be sacrificed under moribund condition during the test period, are necropsies and at the time ofterminal 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 ofthe test item will be retained for archive purpose. Any residual test item will be disposed ofat 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 ofManufacture September 08, 2011 Date ofExpiry September 08, 2013 Appearance Yellow to tan granular 

### Storage Condition (atJRF) As perthe 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 bethe responsibility ofthe Sponsor. JRF 

### Test Item Code SNB444JRF Study Number: 496-1-04-4306 Page 7 of17 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 OperatingProcedures (SOP) Unless otherwise specified all procedures mentioned in the study plan are subject to detailed Standard Operating Procedures ofJai Research Foundation. 3.3 Amendment to StudyPlan 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 sentto 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 offormal study plan amendment. 3.4 Deviation from the StudyPlan Any deviation (unplanned change) from the study plan will be documented in study file and reported in the study report. 3.5 QualityAssurance 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 LaboratoryAnimals Facility' issued by the Committee for the Purpose ofControl and Supervision ofExperiments 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 ofbiological 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 HumaueEndpoint Moribund animals or animals obviously in pain or showing signs ofsevere and enduring distress shall be humanely killed, and are considered in the interpretation ofthe 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 ofthe acclimatization rats will of 10 to I Iweeks old. Care will be taken to avoid mating ofsiblings 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 ofSelection ofTest 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 ofthe 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 ofexperimental room will be swept and all work tops and floor will be cleaned with a disinfectant solution. Detail ofdisinfectant will be document in the raw data and reported in the final report.JRF StudyNumber: 496-1-04-4306 Page 9 of17 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 perhour. 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 ofthe 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 atJai Research Foundation and most recent results will be included in the final report. 5.8 Diet andWater The experimental animals will be fed adlibitum 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 ofanalysis ofnutrient content from the food supplier. Quality offood 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 ofthe treatment, the body weight ofthe rat will not exceed ± 20% ofthe mean body weight in each sex. Rats out ofthis 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 ofthe 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 ofmating. 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 MatingProcedure During the mating period, the male and female rats will be mated in a I: I mating ratio to procure the FI generation. Each morning, the females will be examined for the presence ofsperm 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 ofrats. After evidence ofcopulation, 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. Ifmating has not occurred during two weeks mating period, re-mating of females with proven males ofthe 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. EXPERIMENTALPROCEDURE 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 andMode ofAdmiuistratiou audJustification 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 ofthe test item and will be intimated to the Sponsorthrough a formal study plan amendment.JRF StudyNumber: 496-1-04-4306 Page 11 of17 6.4 Preparation ofDose Formulations Required quantity oftest item for each dose group will be received from TICO. Test item will be formulated in suitable vehicle. The dose formulations (mixture oftest 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 oftheTest Item in the Vehicle The stability ofactive ingredient (a.i.) in vehicle will be determined priorto initiation ofthe study by using validated analytical method. 6.6 Homogeneity and Active Ingredient Concentration ofthe 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 ofDoses 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 ofdose 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 sametime each day. The control group will be a vehicle-control group. The control group will be received the vehicle in the highest volumeused. 6.8 Experimenta1 Schedule Dosing ofboth 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 ofall the females). Females will be further dosed during pregnancy and up to post-partum day 3. 6.9 Summary oftheExperimentalDesign, Group and NumberofAnimals Group Dose Total N° ofRat Minimum N° ofPregnantFemale RatRequire for Study N° (mg/kg b. wt./day) Male Female GI Control (0) 

### 15 15 10 G2 Low Dose 

### 15 15 10 G3 Mid Dose 

### 15 15 10 G4 High Dose 

### 15 15 10JRF 

### StudyNumber: 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 ofreaction to treatment or premature delivery, will be sacrificed and examined as perthe 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 ofthe experiment. 

### 7.1.3 BodyWeights lndividual body weight ofmale and female rats will be recorded on the first day ofdosing 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 ofparturition (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 ofall 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 offeed 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 ofpups, sex ofpups, stillbirths, dead pups and the presence ofgross anomalies. Pups found dead ifnot macerated on the day of littering will be examined for possible defects and cause ofdeath and preserved. 

### 7.2.1 PupBodyWeight 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 ofCO2 when they are not longer for assessment ofreproductive effects. Male rats will be scarified on one day after terminal sacrifice ofall 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 paidto the organs ofthe reproductive system. 7.3.3 During sacrifice, number ofcorpora lutea and implantation sites will be recorded. 

### 7.4 OrganWeigbt At the time ofsacrifice, the testes and epididymides ofall male adult rats will weighed. 

### 7.5 Preservation ofOrgansrrissues At the time of sacrifice, following organs/ tissues ofrats 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 orModified 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 ofother 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 ofspermatogenic cells into the lumen. Examination of the intact epididymis will be included the caput, corpus, and cauda, which can be accomplished by evaluation ofa 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 ofthe primordial follicle population. PAS and hematoxylin staining will be used for examination ofthe male reproductive organs.JRF Study Number: 496-1-04-4306 Page 14 of 17 8. DATA RECORDING AND EVALUATION 8.1 DataRecording 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 ofthis toxicity study will be evaluated in tenns ofthe 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 othertoxic effects. 

### 8.2 DataEvaluation The findings ofthis toxicity study will be evaluated in tenns ofthe 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 othertoxic 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 ofproportions. Significance between control and treatment groups will be calculated with statistical significance of95% confidence level. 9. REPORT Unless otherwise instructed by the Sponsor, one copy ofthe 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 (ifappropriate): Justification for choice ofvehicle ifotherthan water. 3. Test animals: a. Species/strain used. b. Number, age and sex ofanimals. c. Source, housing conditions, diet, etc. d. Individual weights ofanimals at the start ofthe test.JRF Study Number: 496-1-04-4306 Page 15 of17
4. Test conditions: a. Rationale for dose level selection. b. Details oftest item formulation, achieved concentrations, stability and homogeneity ofthe preparation. c. Details ofthe administration ofthe test item. d. Details offood and waterquality. 5. Results (toxic response data by sex and dose): a. Time ofdeath during the study or whether animals survived to termination. b. Nature, severity and duration ofclinical observations (whether reversible or not). c. Body weightlbody weight change data. d. Food consumption, ifapplicable. 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 ofimplantations and number ofcorpora lutea. i. Calculations ofpre- and post implantation loss. J. Detailed description ofhistopathological findings. k. Statisticaltreatment ofresults, where appropriate. 6. Discussion ofresults. 7. Conclusions 8. Signed study plan and study plan amendment(s) (ifany) 9. Record ofdeviation(s) from the study plan (ifany) 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 ofthe final report and the representative sample ofthe test item will be retained in the GLP Archives at Jai Research Foundation for a period oftenyears. Tissues, slides and blocks will be archived for a period offive years. Atthe 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.JRF Study Number: 496-1-04-4306 Page 16 of17 11. REFERENCES 

### 40 CFR 799.5089 Chemical Testing Requirements for Third Group ofHigh 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 ScreeningTest ofSodium 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 VAl"lDERLlP DALTON, GA 30720

Name ofSponsor's Representath:e

Signature and Date

### Page 1of2

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