JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 5 of 217



### CONTENTS

PAGE N°

TITLE PAGE

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

STATEMENT OF GLP COMPLIANCE

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

STATEMENT OF COMPLIANCE WITH GOOD LABORATORY PRACTICE

.............................. 3

STAT EMENT OF QUALITY ASSURANCE

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

CONTENTS

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

PERSONNEL INVOLVED IN THE STUDY

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

LIST OF ABBREVIATIONS

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

SUMMARY

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

1. INTRODUCTION

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

1.1 Study Objective

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

1.2 Regulatory Guideline s

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

1.3 Test Method Principle

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

1.4 Test Facility and Study Period

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

1.5 Archives

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

2. TEST ITEM

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

3. MATERIAL

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

3.1 Instrument s and Equipment

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

3.2 Solvent and Chemical

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

3.3 Vehicle

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

4. ANIMAL WELFARE

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

5. TEST SYSTEM

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

5.1 Test System Specification

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

5.2 Justification for Selection of the Test System

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

5.3 Location of the study

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

5.4 E xperimenta l Room Sanitation

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

5.5 Acclimatization

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

5.6 Environmental Conditions

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

5.7 Housing

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

5.8 Diet and Water

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

5.9 Grouping

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

5.10 Animal Identification

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

5.11 M ating Procedure

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

6. EXPERIMAENTAL PRO CEDURE

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

6.1 Vehicle and Justification for Choice

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

6.2 Route and Mode of Test Item Administration

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

6. 3 Dose Level and Justification

.......................................................................................... 18JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 6 of 217

CONTENTS (Continued)

6.4 Preparation of Dose Formulations

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

6.5 Stability of the Test Item in the Vehicle

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

6 .6 Homogeneit y and Active Ingredient Concentration of the Test Item in the Vehicle

..... 18

6.7 Administration of Doses

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

6.8 Experimental Schedule

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

6.9 Experimental Outline

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

7. OBSERVATIONS

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

7. 1 Parental Rat

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

7.2 Pups

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

7.3

. Necropsy

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

7.4 Organ Weight

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

7.5 P reservation of Organs/Tissues

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

7.6 Histopathology

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

8. I NDICES CALCULATION................................................................................................... 21

9. DATA RECORDING AND EVALUTION

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

9.1 Data Recording

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

9.2 Data Eval uation

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

10. RESULTS AND DISCUSSION

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

10.1 Parental Data

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

10. 1.1 Mortality

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

10.1.2 Cli nical observations

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

10.1.3 Body weight and Body Weight Change

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

10.1.4 Food Consumption

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

10.1.5 Fertility Data

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

10.2 Pup Data 24

10.2.1 Mortality

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

10.2.2 Clinical Sign

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

10.2.3 Body Weight

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

10.2.4 Live Birth and Survival Inde x

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

10.2.5 Litter Size and Sex Ratio

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

10.3 H omogeneity and Active Ingredient Concentration of the

Test Item in the Vehicle

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

10.4 Pathology Report

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

10. 4 .1 Organ Weight

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

10.4.2 Ma croscopic Findings

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

10. 4.3 Microscopic Findings

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

1 1. CONCLUSION

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

12. REFER E NCES....................................................................................................................... 27JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 7 of 217

CONTENTS (Continued)

TABLES

1. Experimental Summary

................................................................................................ . 28

2. Summary of Clinical Symptoms

– Male (P)

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

3. Summary of Clinical Symptoms

– Femal e (P)

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

4. Summar y of Weekly Body weight (g)

– Male (P)

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

5. Summary of Weekly Body weight (g)

– Female (P)

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

6. Summary of Weekly Body Weight Change (%)

– Male (P)

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

7. Summary of Weekly Body Weight Change (%)

– Female (P)

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

8. Summary of Cage wise F oo d Consumption (g)

– Male (P)

........................................... 35

9. Summary of Cage wise F oo d Consumption (g)

– Female (P)

....................................... 36

10. Summary of Number of Pups (F1)

................................................................................. 37

11. Summary of Weight (g) of Pups (F1)

............................................................................ 38

12. Summary of Effect on Fertility, Reproduction and Development

................................ . 39

APPENDICES

1. Clinical Sym ptoms of Individual Male Rat (P)

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

2. Clinical Symptoms of Individual Female Rat (P)

.......................................................... 47

3. Weekly Body Weight (g) of Individual Male Rat (P)

.................................................... 59

4. Weekly Body Weight (g) of Individual Female Rat (P)

................................................ 63

5. Weekly Body Weight Change (%) of Individual Male Rat (P)

..................................... 67

6. We ekly Body Weight Change (%) of Individual Female Rat (P)

.................................. 71

7. F oo d Consumption (g) of Individual Male Rat (P)

........................................................ 75

8. F oo d Consumption (g) of Individual Female Rat (P)

.................................................... 79

9. Pre

- coital Interval and Duration of Gestation Da ys of Individual Female Rat (P)

........ 83

10. Clinical Symptoms of Individual Pups

.......................................................................... 87

11. Litter Size (N°) and Sex Ratio (M : F) of Individual Female

...................................... 10 0

12. Body Weight (g) of Individual Pup s

............................................................................ 10 4

13. Mean Pup ’ s Body Weight (g) of Individual Female

.................................................... 117

14. Pre and Postnatal D ata

................................................................................................ . 121

15. Dose Formulation Analysis Report

.............................................................................. 12 5

16. Patho logy Report

......................................................................................................... 141

17. Analytical Report on Residues of Organochloride and Organophos phorus

pesticides and Heavy Metals in F oo d, W ater and Paddy Husk

................................ .... 183

1 8. Result of Microbiological Analysis of Rat Pellet F oo d, Water and Paddy Husk

........ 186

19. Nutrient Analysis Report of Rat Pellet F oo d

............................................................... 190

20. Signed Study Plan and Study Plan Amendment s

......................................................... 191

21. Record of Deviation s from Study Plan

........................................................................ 21 5

22. Certificate of Analysis of sodium 3

- nitrobenzene sulfonate

........................................ 21 6

23

. G LP Endorsement of Compliance

............................................................................... 21 7JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 9 of 217



### LIST OF ABBREVIATIONS

BMR

- Barrier Maintained Rodent

b. wt.

- Body W eight

F

- Female

g - G ram

G

- Group

G1

- Control (R everse O smosis water )

G2

- Low dose ( 10 mg /kg b

. wt./day )

G3

- Mid dose (1 00 m g /kg b. wt. /day )

G4 - H igh dose ( 10 00 m g /kg b. wt./day )

JRF

- Jai Research Foundation

- - Not Applicable

M - Male

mL

- Millilitre

mg - Milligram

NA

- Not Applicable

N

- Number of A nimals / O bservations

N°

- Number

n - Number of Cages

NAD

- No A bnormalities D etected

OECD

- Organisation for Economic Co

- operation and Development

P

- Parental Animals

PT

- Pre

- exposure /Pre

- treatment

SD

- Standard D eviation

TOX

- Department of Toxicology

TS

- Terminal S acrifice

TI CO

- Test I tem Control Office

TI DS

- Test Item Data Sheet

%

- Percent

°C

- Degree C entigrade

Note: Sodium 3

- nitrobenzene sulfonate (CAS # 127

- 68

- 4; US EPA HPV 3) is represented as Sodium

3- nitrobenzene sulfonateJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 10 of 217



### SUMMARY

Methods

Th e present study was conducted to obtain initial information on effects of Sodium 3

- nitrobenzene

sulfonate on reproduction and/or development when administered through oral gavage to male and female

Wistar rats prior to mating, during mating , post

- mating and gestation periods , and until p ost

- partum day

3

. The methods followed were based on the g uidelines of the OPPTS 870.3550, ( July

, 2000 ).

Total 60 male and 60 female Wistar rats were randomly divided into four groups

. Three dose levels of

sodium 3

- nitrobenzene sulfonate were used and do ses tested were 10 (G2

- Low dose), 1 00 (G3

- Mid dose)

and 1000 (G 4- High dose) mg /kg b

. wt ./ day

. A concurrent control group of rats received vehicle only

(R everse O smosis water ).

All rats were observed twice a day for clinical signs and mortality and morbidi ty throughout the treatment

period. Dose formulation samples were analyzed for A.I

. and homogeneity twice during study. Body

weight of individual male animal were determined weekly throughout treatment period. Food

consumption of individual male animal was determined weekly during pre

- mating and post

- mating

period

. Body weight and food consumption of female animals were determined weekly prior to mating,

and on gestation days 0, 7, 14, 20, within 24 hours of parturition and post

- coitum day 25. Dam and pup’s

body weight were recorded on lactation day s 0 and 4.

A t the end of the treatment period, male and female rats were sacrificed by carbon dioxide asphyxiation

and subjected to gross pathological examination and the organs/tissues defined in study plan were

removed, weighed and processed for histopathological evaluation. Pups were sacrificed by

intraperitoneal ( i.p ) administration of sodium thiopentone

. Absolute organ weights were recorded and

relative organ weight was calculated for the organs defined in st udy plan

. Numbers of corpora lutea and

implantation sites were recorded in dams. Testes, epididymis in male and ovary in female rats of control

and high dose groups were examined microscopically

.

Results

No clinical sign and mortality wa s observed in paren tal animals.

The dose formulation analysis result revealed that the mean calculated concentration was within the

acceptance level of ± 10% of nominal concentration and CV% was < 10% which indicates that the dose

formulation of sodium 3

- nitrobenzene sulfon ate in reverse osmosis was homogeneous and the

concentration was within the acceptance level.

No t reatment related change was observed in body weight, food consumption and litter size in test dose

groups as compared to control group. D uration of gestation, fertility index, pregnancy rate and parturition

index in all the treatment groups was comparable with concurrent control group. No treatment related pre

implantation loss, post

- implantation loss and post

- natal loss was observed in any of the groups treate d

with sodium 3- nitrobenzene sulfonate as compared to control group.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 11 of 217

No treatment related clinical sign and mortality was observed in pups. No treatment related

macroscopic/microscopic finding was observed across the groups.

Conclusion

On the basis of the results obtained in the present study

, it is concluded that sodium 3- nitrobenzene

sulfonate did not produce adverse effect in reproduction and development up to the dose level of 1 00 0

mg/kg b. wt./day when administered through oral gavage in Wistar rats p rior to mating, during mating,

post

- mating, gestation period and until post

- partum day 3.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 12 of 217



### 1. INTRODUCTION

1.1 Study Objective

This study was conducted to determine the initial information on reproduction and/or

development occurring as a result of daily repeated administration of sodium 3- nitrobenzene

sulfonate through oral gavage in Wistar rats prior to mating, during mating

, post

- mating,

gestation period and until post

- partum day 3

.

1.2 Regulatory Guideline s

This study was conducted according to :

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

OPPTS (OCSPP) 870.3550, EPA712 –C– 00

– 367, July 2000. “Reproduction/Developmental

Toxicity Screening Test ”

a nd

40 CFR 799.9355, December 15, 2000. “TSCA Reproduction/Develop mental Toxicity Screening

Test”

1.3 Test Method Principle

The test item, sodium 3- nitrobenzene sulfonate was administered orally by gavage to 3 groups of

male and female rats. A concurrent control group of male and female rats were given reverse

osmosis wa ter only

. Male rats were dosed for two weeks prior to mating, during mating and up to

o ne day after terminal sacrifice of all the females

. Female rats were dosed for two week s prior to

mating, variable time to conception , during gestation period and up to post

- partum day 3.

1. 4 Test Facility and Study Period

The study was performed at the Department of Toxicology, Jai Research Foundation, Valvada

-

396 108, Dist. Valsad, Gujarat, India.

Study Initiation : February 24 , 2012

Experiment Start : Decembe r 04 , 2012

Treatment Start : December 11 , 2012

Cohabitation Start : December 25 , 2012

Terminal Sacrifice

Parental Male : January 28

, 2013

Parental Female with Pups : From January 2 1 , 2013 to January 27, 2013

Experiment Completion : April 19, 2013

S tudy Completion : June 14, 2013JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 13 of 217

1. 5 Archives

All original raw data including any storage medium for electronically recorded data,

documentation, the signed study plan, the study plan amendment s

, the draft report, a copy of the

final report , tissues, blocks and slides and representative sample of the test item will be retained

in the GLP archives at JRF for a period of ten years. At the end of this period, the Sponsor's

instructions will be sought to either extend the archiving period or return the archived material to

the Sponsor or for the material to be disposed of

.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 14 of 217



### 2. TEST ITEM

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

Test Item Name : Sodium 3

- nitrobenzene sulfonate

IUPAC Name : Benzenesulfonic acid, 3- nitro

- , sodium salt (1:1)

CA Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CAS N° : 127

- 68 -4

Batch N° : 50623

Purity : 96.9% (Refer Certificate of Analysis in Appendix N° 2 2)

Manufactured by : Tiarco Chemical

Supplied by : R eservol LP

Date of Manufacture : September 08, 2011

Date of Expiry : September 08, 2013

Appearance : Yellow to tan granular

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

the test i tem, the test item was stored in its original container as

supplied by the Sponsor at 40

- 90 °F (room temperature) in the

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

storage is the responsibility of the Sponsor.

Data related to the char acterization of the test item were documented in a Certificate of A nalysis

provided by the Sponsor and are included as an appendix (Appendix N° 2 2 ) in the study report.

The test item information and analytical identification provided by the Sponsor were co nsidered

as an adequate description of the characterization, purity and stability of the test item and is the

responsibility of the Sponsor.

3. MATERIAL

3.1 Instruments and Equipment

Tattoo Machine : Animal Identification & Marking System, Inc.

Balance s : 1) Sartorius AG (Capacity

– 0.1 g to 2100 g)

2) Adair Dutt, Semi

- micro Electronic Balance

(Capacity

– 0.1 mg to 210 g)

3) Contech Electronic Weighing Scale

(Capacity

– 10 mg to 210 g)

Magnetic Stirrer : DBK Instruments Pvt. Ltd

.

Syringe and Cann ula : 5.0 mL Borosilicate Glass Syringes, 16 g auze x 5 cm r at

c annula

Microscope : N. K. Jain Instruments Pvt. Ltd

.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 15 of 217

3.2 Solvent and Chemical

Disinfectant Formulation : Dettol Surface Disinfectant Formulation 2.5 %

Reckitt Benckiser (India) Limited

Germ Free Hand Sanitizer

(65% Ethanol in Gel Base) : Median Biopharma

Sodium Chloride (NaCl) : Fisher Scientific

3.3 Vehicle : R everse O smosis Water

4. ANIMAL WELFARE

The study was carried out in compliance with the ‘Guidelines for Laboratory Animal s Facility’

issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals

(CPCSEA), India. These guidelines promote the humane care of animals used in research by

providing specifications that has been enhancing animals well being and experimental quality for

the advancement of biological knowledge that is relevant to humans and animals.

Jai Research Foundation is committed to enhance animal welfare and ensures that studies are

designed and conducted to cause the minimum suffering or distress to the animals consistent with

the scientific objectives and in accordance with Jai Research Foundation’s policy on animal

welfare.

Project proposal for the experimentation subjected to approval by Institutional Animal Ethics

Committee ( IAEC), Jai Research Foundation.

JRF is also accredited with AAALAC (Association for Assessment and Accreditation of

Laboratory Animals Care) that promotes humane care of animals during the experiments.

5. TEST SYSTEM

5.1 Test System Specification

Healthy , young adult rats ( Rattus norvegicus ) of Wistar strain obtained from Animal Breeding

Facility, Jai Research Foundation were used for the study

. Female rats were nulliparous and non

pregnant. At initiation of dosing rats were 10 to 11 weeks old.

5.2 Justif ication for Selection of the Test System

Rat ( Rattus norvegicus ) was selected as a test system because it is a readily available laboratory

rodent species. It has been historically shown as acceptable model for developmental and

reproductive toxicity test ing and recommended by various regulatory authorities. In addition, a

large amount of background knowledge was accumulated on this strain.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 16 of 217

5.3 Location of the Study

During the study period, the test animals were located in the Barrier M aintained R odent F acility

(BMR Room N° 12 ) , Department of Toxicology, JRF

.

5.4 Experimental Room Sanitation

Each day, floor of experimental room was swept and all work tops was cleaned with a

disinfectant formulation . Detail of disinfectant was documented in the raw data and reported in

section 3.2 of th is report.

5. 5 Acclimati z ation

T otal of 60 male and 60 female rats were selected for acclimatization. The rats were received into

the experimental procedure room and acclimati z ed for a period of 7 days prior to randomization

.

Fo r randomiz ation 60 male and 60 female rats were selected for the study

. During this period, rats

were observed once daily for clinical signs and twice daily for mortality and morbidity.

5. 6 Environmental Conditions

Rats were maintained in Barrier Ma intained Rodent Facility in an environment controlled room

(BMR Room N° 12 ). The photoperiod was 12 hours fluorescence lighting and 12 hours darkness;

light hours were 06.00

- 18.00 hours, approximately. Environmental condition during study

period was as g iven in below table:

Environmental

Parameters Temp erature (°C) Relative

H umidity (%) Air C hange

(per hour) Mean Light

Intensity

(LUX) Max imum Min imum Max imum Min imum

December 2012 23 20 67 65 20 189

January 2013 23 20 66 58 19 220

5. 7 Housing

Thro ughout the experiment period, the rats were housed individually, with the exception that

during the mating period, the rats were housed in groups of 2 rats/cage (one male plus one

female). Mated female rat were caged individually and nesting material (shre dded paper) from

14 th day of gestation was provided. Animals were housed in solid floor polypropylene rat cages

(size: 41

. 0 c m x 28

. 2 c m x 1 8.0 c m)

. Each cage was fitted with a stainless steel top grill having

provision for keeping rat pellet food and a po lypropylene water bottle with stainless steel drinking

nozzle. Cages were placed on 5 tier racks. Cages were changed twice a week . The cages and

water bottles were cleaned, dried and then sterilized. After sterilization, they were stored in the

UV room ins ide the Barrier Maintained Rodent Facility till used. The bottom of the cages was

layered with clean sterilised pa ddy husk as a bedding material. Cage rotation was done during

pre

- mating period, once in a week.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 17 of 217

Paddy husk is being routinely analyzed once in six month s interval for commonly found

contaminants and the most recent result are incorporated in the report (Refer Appendi ces 17 and

18 ).

5. 8 Diet and Water

The experimental animals were fed ad libitum with standard rodent pellet diet ( Teklad Certifie d

Global 16% Protein Rodent diet manufactured by Harlan , USA ) with an unlimited supply of

clean and filtered drinking water ( filtered through reverse osmosis water filtration system) in

autoclave sterilised polypropylene bottles.

Quality of food and water (microbial and chemical contaminant analysis) is being checked and

monitored at every six months interval in JRF. Every food consignment is subject to microbial

examination in JRF. The most recent analysis reports are presented in the report ( Refer

Append i ces 17 , 1 8 and 1 9 ).

5. 9 Grouping

Prior to randomi z ation, the rat s were weighed. H ealthy rats were allocated to the study. Rats were

randomly allocated to different groups using an in

- house developed validated computer software

program (Gad and Weil, 1994 )

. Animals were divided into f our groups [G1 (control), G2 (low

dose), G3 (mid dose), G4 (high dose) ]. At the start of the treatment, the body weight variations

among the animals were within ± 20 % of the mean body weight for each sex.

5.10 Animal Identifi cation

During acclimatization, a temporary animal number were marked with indelible non

- toxic marker

pen on the dorsal surface of the tail. After grouping, i ndividual animal was identified with

individual unique number tattooed at the base of tail and each cage was labeled with a colored

cage card showing Study N°, Test Item Code, Group N° & Sex, Dose (mg/kg b. wt./day) , Study

Code, Cage N° and Animal N°. Pups were marked with indelible non

- toxic marker pen.

5. 11 Mating Procedure

During the mating period, t he male and female rats were mated in a 1:1 ratio to procure the F1

generation. Each morning, the females were examined for the presence of sperm and the ‘day 0’

of pregnancy was recorded. ‘Day 0’ of pregnancy is defined as the day on which sperms are

obse rved in the vaginal smears of rats. After evidence of copulation, mated female rats were

caged individually.

For each mating, each female was placed with a single male from the same dose level (1:1

mating) until copulation occurs. Detail record of the mat ing pairs was maintained in the raw data.

Care was taken to avoid mating of siblings.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 18 of 217

6. EXPERIMENTAL PROCEDURE

6.1 Vehicle and Justification for C hoice

Reverse Osmosis (RO) water was selected as a vehicle based on solubility check performed at

JRF and af ter consultation with the sponsor

.

6.2 Route and Mode of Test Item Administration

The oral route of administration has been selected for this study since it is an exaggerated model

of the reasonably foreseeable exposure in human

.

6.3 Dose Level and Justifi cation

D ose levels were selected after consultation with Sponsor based on d ose range finding study “JRF

Study N° 410 -1- 04

- 5132”. In this study, three dose level s i.e., 10, 100 and 1000 mg/kg b. wt./day

were tested . A t each dose level no treatment related toxic effects were observed as compared to

control group.

Based on this d ose levels selected w ere 0 (G1

- Control), 10 (G2

- Low), 1 00 (G3

- Mid) and 1000

(G4

- High) mg/kg b. wt./day.

6. 4 Preparation of Dose Formulations

T est item was dissolved in RO Water to obtain desired concentrations. The t est formulation s were

prepared every day prior to dosing and used within 2 hours. Prior to dosing, t est formulation was

thoroughly mixed using magnetic stirrer and during the administration, they were mixed

continuously with glass rod/spatula in order to maintain homogeneity.

6.5 Stability of the Test Item in the Vehicle

The stability of active ingredient (a.i.) in vehicle was determined in Study N° 228 -2- 13

- 5312 by

using a 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 were analyzed before start and once during the treatment period from the main study.

Result o f dose formulation analysis is appended in A ppendix N° 15.

6. 7 Administration of Doses

Test formulations and vehicle were administered to male and female rats daily for seven days a

week. The dose volume for the administration was 10 mL/kg body weight. The doses were

adjusted according to the most recent ly recorded body weight. The control group animals were

given vehicle only . First day of dosing were designated as d ay 1 for each animal

.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 19 of 217

6. 8 Experimental Schedule

Dosing of both the sexes were initiated 2 weeks prior to mating and continued during the mating

period. After mating, the male rats were further dosed up to and including the day before

scheduled sacrifice ( o ne day after terminal sacrifice of all the females). Females were further

dosed during pregnancy and up to post

- partum day 3.

6.9 Experimental Outline

Group

N° Group Dose

(mg/kg b. wt

. /day ) N° of Animals Animal ID N°

M F M F

G1 Vehicle Control 0 15 15 1- 15 16

- 30

G2 Low Dose 10 15 15 31

- 45 46

- 60

G3 Mid Dose 100 15 15 61

- 75 76

- 90

G4 High Dose 1000 15 15 91

- 105 106

- 120

7. OBSERVATIONS

7.1 Parental Rat

7.1.1 Mortality and Morbidity

Rats were observed twice a day for mortality and morbidity. P up, which die d during study, were

weighed and subjec ted to post

- mortem examination.

7.1.2 Cl inical Signs

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

behavior pattern were noted and recorded twice a day, every day throughout the period of the

treatment

.

7.1.3 Body Weights

Individual body weight of mal e and female rats were recorded on the first day of dosing and at

weekly interval thereafter. During gestation period, females were weighed on gestation days 0, 7,

14 and 20 and within 24 hours of parturition (day 0 or 1 post

- partum), and post

- partum 4 (i. e

.,

lactation day 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 were recorded.

7.1.4 Food Consumption

During pre

- mating, gestation and lactation periods food consumption were measured for females

on same day when body weight recorded

. For males, food consumption w as measured weekly

during pre

- mating and post

- mating period

.

Food consumption was not measured during cohabitation period.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 20 of 217

7.2 Pups

Each litter w as examined after delivery to establish the number of pups, sex of pups, stillbirth

,

dead pups, runts (pups that are significantly smaller than corresponding control pups) and the

presence of gross anomalies. P ups found dead on the day of littering were examined for possib le

defects and cause of death.

7.2.1 Pup Body Weight

The total weight of pups in each litter were calculated (by summing the individual pup body

weights for each litter and litter weights were statistically analyzed to determine treatment

effects ). Indiv idual pup body weight w as recorded on lactation days 0 and 4.

7.3 Necropsy

All the surviving rats were sacrificed by using over dose of C O 2 and live pups were sacrificed by

intraperitoneal injection of sodium thiopentone ( barbiturates )

. Male rats were sa crificed one day

after terminal sacrifice of all the females

. Female rat along with the pups were sacrificed on post

partum day 4. Females which have not delivered on day 25 post

- coitum were sacrificed on day 25

post

- coitum.

At the time of sacrifice or de ath during the study, all pups were examined macroscopically for

any structural abnormalities or pathological changes. Special attention paid to the organs of the

reproductive system.

During sacrifice, number of corpora lutea and implantation sites was re corded in dams

.

7.4 Organ Weight

At the time of sacrifice, the testes and epididymis of all male adult rats we re weighed.

7.5 Preservation of Organs/Tissues

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

me dium;

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

(preserved in 10% n eutral b uffered formalin)

Ovaries, test e s and epididymis (preserved in Bouin’s fixative)

Organs with gross lesion (preserved in 10% n eutral b uf fered formalin)

7.6 Histopathology

Detailed histological examination w as performed on the ovaries, testes and epididymides of the

rats from the highest dose group and the control group.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 21 of 217

Detailed testicular histopathological examination (e.g., using Bouin’s fixative, paraffin

embedding and transverse sections of 3-6μ m thickness) were conducted with special emphasis on

stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation

should identif y treatment related effe cts 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 includes the caput, corpus and cauda, which can be accomplished by

evalu ation of a longitudinal section. The epididymis were evaluated for leukocyte infiltration,

change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. Periodic Acid

Schiff ( PAS ) and hematoxylin staining were used for examination of the male reproductive

organs. Histopathological examination of the ovary was detected for qualitative depletion of the

primordial follicle population.

8. INDICES CALCULATION

1. Pre

- implantation loss:

2. Post

- implantation loss:

3. Mating index:

4. Ferti lity index:

5. Gestation index:

6. Live birth index:

7. Viability index on day 4 post

- partum: 

### 100

ons implantati of Number concepti live of Number

- sites on implantati of Number

100

animals paired of Number animals mated of Number 100

lutea corpora of Number sites on implantati of Number

- lutea corpora of Number

100

females pregnant of Number pupsborn live with females of Number

100

pups delivered of Number pupsborn live of Number

100 pupsborn live of Number partum -post 4 day on pups surviving of Number 100 pairs mated of Number partners female pregnant of NumberJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 22 of 217

9. DATA RECORDING AND EVALUATION

9 .1 Data Recording

All study observations were recorded in standard formats. Individual animal data were provided.

Additio nally, all data is summarized in tabular form.

9 .2 Data Evaluation

The findings of this toxicity study were evaluated in terms of the observed effects, macroscopic

and microscopic findings. The evaluation were 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 abnormalities, affected reproductive and litter performance, body weight changes, effects

on mortality and any other toxic effects. Statistic al analyses on pup data (e.g., number of pups

born, pup body weights, pup survival) we re conducted on a per

- litter basis.

Non

- pregnant female rats were not considered for statistical analysis. The test parameters we re

analyzed using appropriate statistical techniques using Bartlett’s test, ANOVA and Dunnett’s ‘t’

test, Student's t

- test, Chi

- square test and t est of significance for difference of proportions.

Significance between control and treatment groups was calculated with statistical significance of

95 and 99% confidence level.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 23 of 217



### 10. RESULTS AND DISCUSSION

10 .1 Parental Data

10 .1 .1 Mortalit y (Refer Table 1)

No mortality was observed in animals treated with sodium 3

- nitrobenzene sulfonate or given

reverse osmosis water

.

10 .1 .2 Clinical Observations (Refe r Table s 2 and 3; Appendi ces 1 and 2)

No toxic clinical signs were observed in animals given reverse osmosis water only and treated

with sodium 3

- nitrobenzene sulfonate

.

10 .1.3 Body Weight and Body W eight Change (Refer Tables 4, 5, 6 and 7; Appendices 3, 4 , 5 and 6)

No change in body weight during treatment period, in male and female rats was observed in

sodium 3

- nitrobenzene sulfonate treated groups as compared with control group

.

In male rats, s tatistically significant decrease in body weight change was observed during

treatment days 8

- 15, 29

- 36 and 1

- 43 at 10 mg/kg b. wt./day as compared to control groups.

Statistically significant decrease in body weight change was observed during treatment days 8

- 15,

15

- 22, 29

- 36 and 1

- 43 at 100 mg/kg b. wt./day as compared to control groups, and transient

inc rease during 36

- 43 was observed

. Statistically significant decrease in body weight change was

observed during treatment days 8

- 15 and 29

- 36 at 1000 mg/kg b. wt./day as compared to control

groups.

In female rats, no treatment related change in percent body weight change was observed in all test

dose levels as compared to concurrent control group except transient increase during gestation

period 14

- 20 at 1000 mg/kg b. wt./day.

Rats belonging to different test dose lev els showed inconsistent decrease in percent body weight

change as compared to control group . D ecrease in percent body weight change lacks dose

dependen cy and it lacks concurrent change in food consumption hence, difference in percent

body weight change is considered as non

- treatment related

.

10 .1.4 Food Consumption (Refer Table s 8 and 9; Appendi ces 7 and 8 )

F ood consumption of male and female animals from treatment groups was comparable as

compared to concurrent control group.

10 .1.5 Fertility Data (Refer Table 1 2 ; Appendi ces 9, 13 and 14 )

Male fertility index, female fertility index and gestation index were comparable between control

and all of the treated groups.

Pregnancy rate and parturition index were unaffected by the treatment.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 24 of 217

The duration of the gestation period and pre

- coital interval was comparable among all the dose

groups with concurrent control group

.

No treatment related difference was observed in pre

- implantation, post

- implantation and post

natal loss in sodium 3

- nitrobenzene sulfon ate treated groups as compared to control group.

10 .2 Pup Data

10 .2 .1 Mortality (Refer Table 1 2)

No treatment related difference in pup mortality during the lactation period for male, female and

total (male + female) pup was observed in all sodium 3

- nitro benzene sulfonate treated groups

when compared with control group

.

10 .2 .2 Clinical Sign (Refer Appendix 10 )

No treatment related toxic clinical sign s ( except pup N° 7 of dam N° 46 and 79 showed

weakness ) w ere observed in pups belonging to all groups.

10 .2.3 Body Weight (Refer Table 11 ; Appendices 1 2 and 1 3)

No significant difference in pups body weight was observed in male and female pups of sodium

3- nitrobenzene sulfonate treated groups as compared to control group.

10 .2.4 Live Birth and Survival Index ( Refer Tables 10 , 11 and 12 )

Mean number of live pups, live birth index and pups survival index were comparable between all

treatment groups and the control group

.

10 .2.5 Litter Size and Sex Ratio (Refer Tables 10 and 12 ; Appendi x 11)

Mean male, female an d total (male + female) litter size and sex ratio were not affected in any of

the dose groups

.

10.3 Homogeneity and Active Ingredient Concentration of the Test Item in the Vehicle (Appendix

15)

Before start of the treatment, the mean percentage recovery obtained for test item in vehicle was

96.21

, 96.40 and 96.51 % at 10, 100 and 100 0 mg/kg b

. wt

. /day dose level s , respectively. The

mean percentage recovery obtained during treatment period, for test item in vehicle was 98.49,

98.04 and 97.04% at 10, 10 0 and 10 00 mg/kg b . w t. /day, respectively.

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

level of ± 10% of nominal concentration and CV% was < 10% which indicates that the dose

formulation of sodium 3

- nitrobenzen e sulfonate in reverse osmosis was homogeneous and the

concentration was within the acceptance level.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 25 of 217

10. 4 Pathology Report (Appendix 1 6)

10. 4 .1 Organ Weight

The terminal body weight and organ weight (both absolute and relative weight of testes and

epid idymis) in male rats belonging to treatment groups were comparable to the control group

.

10.4.2 Macroscopic Findings

Parental Rats

External and internal examination of the terminally sacrificed rats of all the groups did not reveal

any lesion of pat hological significance.

Pups

External examination of the terminally sacrificed as well as deceased pups belonging to different

experimental groups did not reveal any lesion of pathological significance

. Internal examination

of the terminally sacrificed pups of different groups did not reveal any lesion of pathological

significance.

10. 4.3 Microscopic Findings

Microscopic examination revealed varying degree of pathological changes in testes and

epididymis of control and high dose groups

. Lesions observe d were spontaneous and hence not

considered as treatment related.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 26 of 217



### 11. CONCLUSION

On the basis of the results obtained in the present study

, it is concluded that sodium 3

-

nitrobenzene sulfonate did not produce adverse effect in reproduction and developme nt up to the

dose level of 1 00 0 mg/kg b. wt./day when administered through oral gavage in Wistar rats prior

to mating, during mating, post

- mating, gestation period and until post

- partum day 3

.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 27 of 217

12

. 

### REFERENCES

40 CFR 799.9355, December 15, 2000 : “TSCA Repro duction/Developmental Toxicity Screening

Test”

.

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

Chemicals (HPV 3).

A. Shrimali , 2012: JRF Study N° 228 -2- 13

- 5312, “ Validation of Analytical Method for

Determination of Sodium 3

- nitrobenzensulfonate Active Ingredient Concentration in Dose

Formulation ”.

D. Ujawane, 2012: JRF Study N° 410 -1- 04

- 5132, Repeated Dose 14

- Day Oral Toxicity Study of

Sodium 3

- nitrobenzensulfonate in Wistar Rats (Dose Range Finding Study)”.

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

Research. 6 th 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, 3 rd Edition, Hayes A.W. (Ed), Raven press Ltd., New York, p p . 221

- 274.

OECD, 1998: OECD Series on Principles of Good Laboratory Practice and Compliance

Monitoring, Number 1, “OECD Principles on Good Laboratory Practice”

ENV/MC/CHEM(98)17 (as revised in 1997).

The Organization for Economic Co

- operation and Development (OECD) Guidelines for the

testing of chemicals, proposal for updating guideline 421 “ Reproduction/Develo pmental Toxicity

Screening Test”

, a dopted by the Council on 27th July 1995.

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

OPPTS (OCSPP) 870.3550, EPA712 –C– 00

– 367, July 2000. “Reproduction/Developmental

Toxicity Screening Test ”.

### JRF Study Number: 496-1-04-4306 (Final Report) Page 28 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 1

Experimental Summary

Group and Dose: G1 - 0; G2- 10; G3 - 100; G4 - 1000 mg/kg body weight/day

N° of female rats kept for cohabitation = 15 per group

Parameters G1 G2 G3 G4

N % N % N % N %

Mortality During Experiment 0 0 0 0 0 0 0 0

Rat Sacrifice Under Moribund Condition 0 0 0 0 0 0 0 0

Pre

- mating and Mating Period

N° of Male 15

- 15

- 15

- 15

-

N° of Female 15

- 15

- 15

- 15

-

N° of Female Housed with Male 15

- 15

- 15

- 15

-

Female Humanely Sacrificed

after Mating 0

- 0

- 0

- 0

-

N° of E + 

### Females 15 100.00 15 100.00 15 100.00 15 100.00

Gestation Period

Female with premature delivery/ abortion 0

- 0

- 0

- 0

-

Non

- pregnant or Females sacrificed at term

(at 25 th 

### day of Gestation) 4 26.67 1 6.67 5 33.33 5 33.33

N° of Females giving birth 11 73.33 14 93.33 10 66.67 10 66.67

N° of Females giving birth to viable pups 11 100.00 14 100.00 10 100.00 10 100.00

N° of Females giving birth to all viable pups 8 72.73 13 92.86 9 90.00 9 90.00

Lactation Period

N° of Female Sacrificed on Lactation Day 4 11 100.00 14 100.00 10 100.00 10 100.00

N° of Female (along with pups) Sacrificed on

Lactation Day 4 10 90.91 14 100.00 10 100.00 10 100.00

Key: G = Group, N = Number of observation, - = Not applicable, E + 

### = Estrus positive and % = Percent

### JRF Study Number: 496-1-04-4306 (Final Report) Page 29 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 2

Summary of Clinical Symptoms

– Male (P)

Group and Dose: G1 - 0; G2 - 10; G3 - 100; G4 - 1000 mg/kg body weight/day

Number of Observations (Rats /group): 15

Symptoms Observed

Clinical Observations N° of Animals

G1 G2 G3 G4

Normal 15 15 15 15

### JRF Study Number: 496-1-04-4306 (Final Report) Page 30 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 3

Summary of Clinical Symptoms

– Female (P)

Group and Dose: G1 - 0; G2- 10; G3 - 100; G4

– 1000 mg/kg body weight/day

Number of Observations (Rats/group): 15

Symptoms Observed

Clinical Observations N° of Animals

G1 G2 G3 G4

Pre

- mating Period

Normal 15 15 15 15

Gestation Period

Normal 15 15 15 15

Lactation Period

Normal 11 14 10 10

Note: Rat N° 21, 25, 26 & 28 (G1); 56 (G2); 76, 77, 80, 87 & 83 (G3) and 106, 110, 113, 114 & 12 0

(G4) were non -pregnant

### JRF Study Number: 496-1-04-4306 (Final Report) Page 31 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 4

Summary of Weekly Body Weight (g)

– Male (P)

Group and Dose: G1 - 0; G2- 10; G3 - 100; G4

– 1000 mg/kg body weight/day

Treatment

Days Group N°

G1 G2 G3 G4

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

1 315.75 21.85 15 314.71 20.49 15 314.90 19.42 15 313.71 19.28 15

8 340.31 23.29 15 338.34 21.68 15 339.51 21.70 15 340.67 22.61 15

15 366.48 26.62 15 358.88 21.77 15 358.21 24.43 15 360.45 22.59 15

22 385.35 29.89 15 373.13 21.46 15 369.26 25.39 15 374.66 21.96 15

29 404.98 33.24 15 388.15 22.30 15 382.81 29.92 15 392.15 23.89 15

36 425.20 38.12 15 402.86 23.36 15 396.88 32.13 15 406.30 25.34 15

43 437.43 43.12 15 415.15 23.70 15 412.09 33.84 15 418.03 27.09 15

Key: N = Number of observations, SD = Standard deviation

### JRF Study Number: 496-1-04-4306 (Final Report) Page 32 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 5

Summary of Weekly Body Weight (g)

– Female (P)

Group and Dose: G1 - 0; G2 - 10; G3 - 100; G4 - 1000 mg/kg body weight/day

Pre-mating Period

Pre

- mating

Days Group N°

G1 G2 G3 G4

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

1 216.99 14.08 15 216.83 13.97 15 217.31 15.36 15 216.91 13.76 15

8 227.93 14.71 15 227.10 14.62 15 229.06 16.50 15 227.23 14.99 15

15 237.45 14.72 15 238.16 15.20 15 236.50 15.65 15 238.05 15.18 15

Gestation Period

Gestation

Day Group N°

G1 G2 G3 G4

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

0 242.47 14.40 11 241.53 16.25 14 241.62 16.94 10 238.53 21.68 10

7 267.94 17.16 11 265.58 16.47 14 266.04 15.75 10 261.04 22.08 10

14 296.55 19.79 11 295.21 17.90 14 295.52 17.84 10 289.43 16.90 10

20 351.45 21.96 11 354.74 22.11 14 351.09 23.09 10 357.05 20.97 10

Lactation Period

Lactation

Day Group N°

G1 G2 G3 G4

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

0 276.81 33.19 11 266.94 16.82 14 271.29 21.82 10 272.25 12.66 10

4 280.25 28.41 11 274.26 16.67 14 277.65 19.01 10 280.45 14.62 10

Key: N = Number of observations, SD = Standard deviation

### JRF Study Number: 496-1-04-4306 (Final Report) Page 33 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 6

Summary of Weekly Body Weight Change (%)

– Male (P)

Group and Dose: G1 - 0; G2- 10; G3 - 100; G4

– 1000 mg/kg body weight/day

During

Treatment

Days Group N°

G1 G2 G3 G4

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

1

- 8 7.79 1.63 15 7.53 1.70 15 7.82 2.32 15 8.57 1.55 15

8

- 15 7.67 1.71 15 6.10  1.30 15 5.49   1.34 15 5.85   1.73 15

15

- 22 5.12 1.41 15 4.00 1.57 15 3.08   1.09 15 3.97 1.28 15

22

- 29 5.08 1.83 15 4.03 0.96 15 3.62 1.69 15 4.67 1.49 15

29

- 36 4.94 1.53 15 3.79  1.06 15 3.66  1.09 15 3.61  1.21 15

36

- 43 2.81 1.52 15 3.06 0.71 15 3.83  1.16 15 2.88 0.90 15

1

- 43 38.47 8.72 15 32.08  5.50 15 30.90  7.81 15 33.31 4.78 15

Key: N = Number of observations, SD = Standard deviation

Note:  = Significantly higher than control (p  0.05),  = Significantly lower than control (p  0.05),

 

= Significantly lower than control (p  0.01)

### JRF Study Number: 496-1-04-4306 (Final Report) Page 34 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 7

Summary of Weekly Body Weight Change (%)

– Female (P)

Group and Dose: G1 - 0; G2- 10; G3 - 100; G4

– 1000 mg/kg body weight/day

Pre-mating Period

During

Treatment

Days Group N°

G1 G2 G3 G4

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

1

- 8 5.06 1.48 15 4.77 2.40 15 5.41 1.11 15 4.75 1.29 15

8

- 15 4.21 1.83 15 4.89 1.82 15 3.30 1.59 15 4.80 2.10 15

1

- 15 9.49 3.00 15 9.88 2.52 15 8.88 2.05 15 9.77 2.52 15

Gestation Period

During

Gestation

Days Group N°

G1 G2 G3 G4

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

0

- 7 10.51 3.10 11 10.02 2.54 14 10.20 2.67 10 9.51 2.88 10

7

- 14 10.67 1.52 11 11.18 1.74 14 11.09 2.08 10 11.11 3.80 10

14

- 20 18.61 4.41 11 20.18 2.86 14 18.83 4.00 10 23.39  2.75 10

0

- 20 45.04 6.65 11 47.06 6.87 14 45.59 9.06 10 50.23 9.18 10

Lactation Period

During

Lactation

Days Group N°

G1 G2 G3 G4

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

0

- 4 1.48 3.88 11 2.79 2.48 14 2.48 3.35 10 3.03 3.06 10

Key: N = Number of observations, SD = Standard deviation

Note:  = Significantly higher than control (p  0.05),

### JRF Study Number: 496-1-04-4306 (Final Report) Page 35 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 8

Summary of Cage Wise Food Consumption (g) - Male (P)

Group and Dose: G1 - 0; G2- 10; G3 - 100; G4

– 1000 mg/kg body weight/day

Treatment

Days Group N°

G1 G2 G3 G4

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

1

- 8 25.20 1.51 15 25.01 1.86 15 25.29 1.18 15 24.78 2.26 15

8

- 15 25.81 2.87 15 24.60 2.05 15 24.55 2.04 15 25.27 2.86 15

23

- 29 25.58 2.91 15 23.83 1.39 15 24.39 2.81 15 24.58 2.38 15

29

- 36 24.90 3.00 15 23.43 2.06 15 22.50 2.39 15 24.19 2.29 15

36

- 43 24.69 3.12 15 23.25 2.11 15 23.13 2.86 15 23.63 2.51 15

43

- TS 25.91 4.39 15 24.61 2.52 15 24.05 2.45 15 24.90 2.56 15

Key: N = Number of observations, SD = Standard deviation, TS = Terminal sacrifice

### JRF Study Number: 496-1-04-4306 (Final Report) Page 36 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 9

Summary of Cage Wise Food Consumption (g)

– Female (P)

Group and Dose: G1

– 0; G2- 10; G3

– 100; G4

–

1000 mg/kg body weight/day

Pre

- mating

Days Group N°

G1 G2 G3 G4

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

1

- 8 19.25 2.09 15 18.63 1.22 15 18.64 1.43 15 18.01 1.12 15

8

- 15 19.47 2.13 15 18.87 1.02 15 18.42 1.68 15 18.39 1.85 15

Gestation Period

Gestation

Days Group N°

G1 G2 G3 G4

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

0

- 7 20.93 1.83 11 21.20 1.45 14 20.28 1.64 10 19.77 1.58 10

7

- 14 21.75 1.98 11 22.11 1.41 14 22.18 1.20 10 21.54 1.76 10

14

- 20 23.18 3.40 11 23.45 2.48 14 23.22 1.99 10 23.17 1.67 10

Lactation Period

Lactation

Days Group N°

G1 G2 G3 G4

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

0

- 4 23.48 3.02 11 24.07 4.05 14 23.41 4.26 10 26.50 4.32 10

Key: N = Number of observations, SD = Standard deviation,

### JRF Study Number: 496-1-04-4306 (Final Report) Page 37 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 10

Summary of Number of Pups (F1)

Group and Dose: G1

– 0; G2- 10; G3

– 100; G4

–

1000 mg/kg body weight/day

Number of Male Pups

Lactation

Day Groups

G1 G2 G3 G4

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

0 4.36 3.01 11 5.29 2.05 14 5.44 2.51 9 6.50 1.43 10

4 4.27 3.13 11 4.93 2.30 14 5.33 2.69 9 6.20 1.32 10

Number of Female Pups

Lactation

Day Groups

G1 G2 G3 G4

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

0 5.73 4.15 11 6.43 2.34 14 5.70 2.31 10 5.70 1.34 10

4 5.55 4.18 11 6.29 2.23 14 5.40 2.17 10 5.60 1.17 10

Number of Male and Female Pups

Lactation

Day Groups

G1 G2 G3 G4

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

0 10.09 4.68 11 11.71 3.17 14 10.60 4.09 10 12.20 1.69 10

4 9.82 5.00 11 11.21 3.29 14 10.20 4.16 10 11.80 1.55 10

Sex Ratio (M/F)

Lactation

Day Groups

G1 G2 G3 G4

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

0 2.07 3.47 11 0.96 0.71 14 1.04 0.50 9 1.21 0.43 10

Key: N = Number of observations, SD = Standard deviation

### JRF Study Number: 496-1-04-4306 (Final Report) Page 38 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 11

Summary of Weight (g) of Pups (F1)

Group and Dose: G1

– 0; G2- 10; G3

– 100; G4

–

1000 mg/kg body weight/day

Weight of Male Pups

Lactation

Day Groups

G1 G2 G3 G4

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

0 6.08 0.78 11 5.98 0.88 14 6.04 0.64 9 6.06 0.34 10

4 10.25 2.35 10 9.75 1.91 14 10.11 1.60 9 10.07 0.74 10

Weight of Female Pups

Lactation

Day Groups

G1 G2 G3 G4

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

0 5.75 0.83 11 5.70 0.68 14 5.79 0.72 10 5.79 0.37 10

4 9.87 2.49 10 9.26 1.43 14 9.63 1.39 10 9.71 0.91 10

Weight of Male and Female Pups

Lactation

Day Groups

G1 G2 G3 G4

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

0 5.91 0.82 11 5.80 0.72 14 5.92 0.66 10 5.94 0.33 10

4 10.07 2.44 10 9.43 1.55 14 9.93 1.45 10 9.89 0.78 10

Key: N = Number of observations, SD = Standard deviation

### JRF Study Number: 496-1-04-4306 (Final Report) Page 39 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3 -nitrobenzene Sulfonate (CAS # 127-68 -4; US EPA HPV 3) in Wistar Rats

TABLE 12

Summary of Effect on Fertility, Reproduction and Development

Observations Values

Dose (mg/kg body weight/day) 0 10 100 1000

Pairs started (N) 15 15 15 15

Females showing evidence of copulation (N) 15 15 15 15

Females achieving pregnancy (N) 15 15 15 15

Conceiving days 1

– 5 (N) 1 4 15 12 13

Conceiving days 6

– 10 (N) 1 0 3 2

Pregnancy = 21 days (N) 0 0 1 0

Pregnancy = 22 days (N) 6 9 6 6

Pregnancy = 23 days (N) 5 5 3 4

Pregnancy  24 days (N) 0 0 0 0

Female sacrificed on post

- coitum day 25 (N) 4 1 5 5

Dams with live young born (N) 11 14 10 10

Dams with live young at day 4 (N) 10 14 10 10

Corpora lutea (mean  SD) 13.09 

2.21 13.50 

2.10 13.30 

3.37 13.70 

2.26

Implants (mean  SD) 11.00 

4.88 12.36 

2.82 11.50 

3.75 13.00 

2.11

ABNORMAL PUPS

Dams with 0 0 0 0 0

Dams with 1 0 0 0 0

Dams with >2 0 0 0 0

### JRF Study Number: 496-1-04-4306 (Final Report) Page 40 of 217

TABLE 12 (Continued)

Observations Values

Dose (mg/kg body weight/day) 0 10 100 1000

LOSS OF OFFSPRING

Pre

- implantation (corpora lutea minus implantations)

Females with 0 6 5 3 7

Females with 1 1 6 4 1

Females with 2 1 2 0 1

Females with ≥ 3 3 1 3 1

Pre

- natal / Post

- implantation (implantations minus live births)

Females with 0 5 6 5 5

Females with 1 2 7 2 3

Females with 2 4 1 2 1

Females with ≥ 3 0 0 1 1

Post

- natal (live births minus alive at post natal day 4)

Females with 0 9 9 7 6

Females with 1 1 3 2 4

Females with 2 1 2 1 0

Females with ≥ 3 0 0 0 0

FERTILITY DATA

Number of females used 15 15 15 15

Number of males housed with female 15 15 15 15

Number of males impregnating female 15 15 15 15

Number of females with sperm positive vaginal

smear 15 15 15 15

Number of female confirmed pregnant 15 15 15 15

Number of females giving birth 11 14 10 10

Number of females giving birth to viable pups 11 14 10 10

Number of females giving birth to all viable pups 8 13 9 9

Duration of gestation (day) (mean  SD) 22.45 

0.52 22.36 

0.50 22.20 

0.63 22.40 

0.52

Key: - = Not applicable

### JRF Study Number: 496-1-04-4306 (Final Report) Page 41 of 217

TABLE 12 (Continued)

Observations Values

Dose (mg/kg body weight/day) 0 10 100 1000

FERTILITY INDEX

Male Fertility Index 100.00 100.00 100.00 100.00

Female Fertility Index 100.00 100.00 100.00 100.00

Gestation Index 73.33 93.33 66.67 66.67

Parturition Index 73.33 93.33 66.67 66.67

Percentage of Pregnant Animals 73.33 93.33 66.67 66.67

Percentage of Non

- pregnant Animals 26.67 6.67 33.33 33.33

Mating index 100.00 100.00 100.00 100.00

Pre

- implantation loss (%) (mean  SD) 19.65  30.05 8.94  12.84 14.07  17.19 4.79  8.32

Post

- implantation loss (%) (mean  SD) 8.45  9.06 6.18  6.15 9.67  13.54 5.57  6.95

Post

- natal loss (%) (mean  SD) 9.79  30.01 4.78  8.61 3.82  6.83 3.08  3.99

Live Birth and Postnatal Day

Group G1 G2 G3 G4

Sex Male Female Total Male Female Total Male Female Total Male Female Total

Live Birth

Index 

### 96.00 98.44 97.37 98.67 100.00 99.39 100.00 98.28 99.07 98.48 100.00 99.19

Postnatal

Loss 

### 3 3 6 6 2 8 1 4 5 4 1 5

### JRF Study Number: 496-1-04-4306 (Final Report) Page 42 of 217

TABLE 12 (Continued)

Pups Litter Size (Number)

Group G1 G2 G3 G4

Sex Male Female Total Male Female Total Male Female Total Male Female Total

Total N° of Pups on

Day of Littering 50 64 114 75 90 165 49 58 107 66 57 123

Pups Still birth on

Day of Littering 2 1 3 1 0 1 0 1 1 1 0 1

Total N° of Live

Pups on Day of

Littering 48 63 111 74 90 164 49 57 106 65 57 122

Litter Size

on LD 4 47 61 108 69 88 157 48 54 102 62 56 118

Pups Mortality Index

Group G1 G2 G3 G4

Sex Male Female Total Male Female Total Male Female Total Male Female Total

During LD

0 to 4 (N) 

### 3 3 6 6 2 8 1 4 5 4 1 5

0

- 4 (%) 

### 6 4.69 5.26 8 2.22 4.85 2.04 6.9 4.67 6.06 1.75 4.07

Pups Survival Index

Group G1 G2 G3 G4

Sex Male Female Total Male Female Total Male Female Total Male Female Total

Survival

Index on

LD 4 94.00 95.31 94.74 92.00 97.78 95.15 97.96 93.10 95.33 93.94 98.25 95.93

Key: LD = Lactation DayJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 43 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 1

Clinical Symptoms of Individual Male Rat (P)

Group : G1 Dose : 0 mg/kg body weight/day

Rat

N° Observatio n During Treatment Period

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

1 Normal 1 49 49 Normal 1 48 48

2 Normal 1 49 49 Normal 1 48 48

3 Normal 1 49 49 Normal 1 48 48

4 Normal 1 49 49 Normal 1 48 48

5 Normal 1 49 49 Normal 1 48 48

6 Normal 1 49 49 Normal 1 48 48

7 Normal 1 49 49 Normal 1 48 48

8 Normal 1 49 49 Normal 1 48 48

9 Normal 1 49 49 Normal 1 48 48

10 Normal 1 49 49 Normal 1 48 48

11 Normal 1 49 49 Normal 1 48 48

12 Normal 1 49 49 Normal 1 48 48

13 Normal 1 49 49 Normal 1 48 48

14 Normal 1 49 49 Normal 1 48 48

15 Normal 1 49 49 Normal 1 48 48JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 44 of 217

APPENDIX 1 ( Continued )

Group : G2 Dose : 1 0 mg/kg body weight/day

Rat

N° Observation During Treatment Period

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

31 Normal 1 49 49 Normal 1 48 48

32 Normal 1 49 49 Normal 1 48 48

33 Normal 1 49 49 Normal 1 48 48

34 Normal 1 49 49 Normal 1 48 48

35 Normal 1 49 49 Normal 1 48 48

36 Normal 1 49 49 Normal 1 48 48

37 Normal 1 49 49 Normal 1 48 48

38 Normal 1 49 49 Normal 1 48 48

39 Normal 1 49 49 Normal 1 48 48

40 Normal 1 49 49 Normal 1 48 48

41 Normal 1 49 49 Normal 1 48 48

42 Normal 1 49 49 Normal 1 48 48

43 Normal 1 49 49 N ormal 1 48 48

44 Normal 1 49 49 Normal 1 48 48

45 Normal 1 49 49 Normal 1 48 48JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 45 of 217

APPENDIX 1 ( Continued )

Group : G3 Dose : 1 00 mg/kg body weight/day

Rat

N° Observation During Treatment Period

Morning

Observation First

Day Last

Day Total

Days Post Dos ing

Observation First

Day Last

Day Total

Days

61 Normal 1 49 49 Normal 1 48 48

62 Normal 1 49 49 Normal 1 48 48

63 Normal 1 49 49 Normal 1 48 48

64 Normal 1 49 49 Normal 1 48 48

65 Normal 1 49 49 Normal 1 48 48

66 Normal 1 49 49 Normal 1 48 48

67 No rmal 1 49 49 Normal 1 48 48

68 Normal 1 49 49 Normal 1 48 48

69 Normal 1 49 49 Normal 1 48 48

70 Normal 1 49 49 Normal 1 48 48

71 Normal 1 49 49 Normal 1 48 48

72 Normal 1 49 49 Normal 1 48 48

73 Normal 1 49 49 Normal 1 48 48

74 Normal 1 49 49 Norma l 1 48 48

75 Normal 1 49 49 Normal 1 48 48JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 46 of 217

APPENDIX 1 ( Continued )

Group : G4 Dose : 1000 mg/kg body weight/day

Rat

N° Observation During Treatment Period

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day T otal

Days

91 Normal 1 49 49 Normal 1 48 48

92 Normal 1 49 49 Normal 1 48 48

93 Normal 1 49 49 Normal 1 48 48

94 Normal 1 49 49 Normal 1 48 48

95 Normal 1 49 49 Normal 1 48 48

96 Normal 1 49 49 Normal 1 48 48

97 Normal 1 49 49 Normal 1 48 48

98 Norm al 1 49 49 Normal 1 48 48

99 Normal 1 49 49 Normal 1 48 48

100 Normal 1 49 49 Normal 1 48 48

101 Normal 1 49 49 Normal 1 48 48

102 Normal 1 49 49 Normal 1 48 48

103 Normal 1 49 49 Normal 1 48 48

104 Normal 1 49 49 Normal 1 48 48

105 Normal 1 49 49 N ormal 1 48 48JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 47 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 2

Clinical Symptoms of Individual Female Rat (P)

Group : G1 Dose : 0 mg/kg body weight/day

Rat

N° Observation During Treatment Period (Pre

- mating and Mating Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

16 Normal 1 19 19 Normal 1 18 18

17 Normal 1 17 17 Normal 1 16 16

18 Nor mal 1 18 18 Normal 1 17 17

19 Normal 1 18 18 Normal 1 17 17

20 Normal 1 16 16 Normal 1 15 15

21 Normal 1 16 16 Normal 1 15 15

22 Normal 1 18 18 Normal 1 17 17

23 Normal 1 17 17 Normal 1 16 16

24 Normal 1 17 17 Normal 1 16 16

25 Normal 1 16 16 Normal 1 15 15

26 Normal 1 22 22 Normal 1 21 21

27 Normal 1 18 18 Normal 1 17 17

28 Normal 1 18 18 Normal 1 17 17

29 Normal 1 19 19 Normal 1 18 18

30 Normal 1 19 19 Normal 1 18 18JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 48 of 217

APPENDIX 2 (Continued)

Group : G1 Dose : 0 mg/kg body weight/day

Rat

N° Ob servation During Treatment Period (Gestation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

16 Normal 19 42 24 Normal 19 41 23

17 Normal 17 39 23 Normal 17 38 22

18 Normal 18 41 24 Norma l 18 40 23

19 Normal 18 40 23 Normal 18 39 22

20 Normal 16 39 24 Normal 16 38 23

21 Normal 16 41 26 Normal 16 40 25

22 Normal 18 40 23 Normal 18 39 22

23 Normal 17 39 23 Normal 17 38 22

24 Normal 17 39 23 Normal 17 38 22

25 Normal 16 41 26 Normal 16 40 25

26 Normal 22 47 26 Normal 22 46 25

27 Normal 18 41 24 Normal 18 40 23

28 Normal 18 43 26 Normal 18 42 25

29 Normal 19 42 24 Normal 19 41 23

30 Normal 19 41 23 Normal 19 40 22JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 49 of 217

APPENDIX 2 (Continued)

Group : G1 Dose : 0 mg/kg body weight/day

Rat

N° Observation During Treatment Period (Lactation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

16 Normal 42 46 5 Normal 42 45 4

17 Normal 39 43 5 Normal 39 42 4

18 Normal 41 45 5 N ormal 41 44 4

19 Normal 40 44 5 Normal 40 43 4

20 Normal 39 43 5 Normal 39 42 4

21

- - - - - - - -

22 Normal 40 44 5 Normal 40 43 4

23 Normal 39 43 5 Normal 39 42 4

24 Normal 39 43 5 Normal 39 42 4

25

- - - - - - - -

26

- - - - - - - -

27 Normal 41 45 5 Normal 41 44 4

28

- - - - - - - -

29 Normal 42 46 5 Normal 42 45 4

30 Normal 41 45 5 Normal 41 44 4

Note: - = Not Applicable

Key: Rat N° 21, 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 50 of 217

APPENDIX 2 (Continued)

Group : G2 Dose : 10 mg/kg body weight/day

R at

N° Observation During Treatment Period (Pre

- mating and Mating Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

46 Normal 1 16 16 Normal 1 15 15

47 Normal 1 19 19 Normal 1 18 18

48 Norma l 1 16 16 Normal 1 15 15

49 Normal 1 19 19 Normal 1 18 18

50 Normal 1 18 18 Normal 1 17 17

51 Normal 1 16 16 Normal 1 15 15

52 Normal 1 19 19 Normal 1 18 18

53 Normal 1 16 16 Normal 1 15 15

54 Normal 1 17 17 Normal 1 16 16

55 Normal 1 19 19 Normal 1 18 18

56 Normal 1 17 17 Normal 1 16 16

57 Normal 1 19 19 Normal 1 18 18

58 Normal 1 17 17 Normal 1 16 16

59 Normal 1 17 17 Normal 1 16 16

60 Normal 1 17 17 Normal 1 16 16JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 51 of 217

APPENDIX 2 (Continued)

Group : G2 Dose : 10 mg/kg body weight/day

Rat

N° Ob servation During Treatment Period (Gestation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

46 Normal 16 39 24 Normal 16 38 23

47 Normal 19 41 23 Normal 19 40 22

48 Normal 16 39 24 Norma l 16 38 23

49 Normal 19 41 23 Normal 19 40 22

50 Normal 18 41 24 Normal 18 40 23

51 Normal 16 39 24 Normal 16 38 23

52 Normal 19 41 23 Normal 19 40 22

53 Normal 16 38 23 Normal 16 37 22

54 Normal 17 39 23 Normal 17 38 22

55 Normal 19 41 23 Normal 19 40 22

56 Normal 17 42 26 Normal 17 41 25

57 Normal 19 41 23 Normal 19 40 22

58 Normal 17 39 23 Normal 17 38 22

59 Normal 17 39 23 Normal 17 38 22

60 Normal 17 40 24 Normal 17 39 23JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 52 of 217

APPENDIX 2 (Continued)

Group : G2 Dose : 10 mg/kg body weight/day

Rat

N° Observation During Treatment Period (Lactation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

46 Normal 39 43 5 Normal 39 42 4

47 Normal 41 45 5 Normal 41 44 4

48 Normal 39 43 5 Normal 39 42 4

49 Normal 41 45 5 Normal 41 44 4

50 Normal 41 45 5 Normal 41 44 4

51 Normal 39 43 5 Normal 39 42 4

52 Normal 41 45 5 Normal 41 44 4

53 Normal 38 42 5 Normal 38 41 4

54 Normal 39 43 5 Normal 39 42 4

55 Normal 41 45 5 Normal 41 44 4

56

- - - - - - - -

57 Normal 41 45 5 Normal 41 44 4

58 Normal 39 43 5 Normal 39 42 4

59 Normal 39 43 5 Normal 39 42 4

60 Normal 40 44 5 Normal 40 43 4

Note: - = Not Applicable

Key: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 53 of 217

APPENDIX 2 (Continued)

Group : G 3 Dose : 1 00 mg/kg body weight/day

Rat

N° Observation During Treatment Period (Pre

- mating and Mating Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

76 Normal 1 19 19 Normal 1 18 18

77 Normal 1 22 22 Normal 1 21 21

78 Normal 1 21 21 Normal 1 20 20

79 Normal 1 17 17 Normal 1 16 16

80 Normal 1 22 22 Normal 1 21 21

81 Normal 1 18 18 Normal 1 17 17

82 Normal 1 18 18 Normal 1 17 17

83 Normal 1 18 18 Normal 1 17 17

84 Normal 1 18 18 Normal 1 17 17

85 Normal 1 19 19 Normal 1 18 18

86 Normal 1 19 19 Normal 1 18 18

87 Normal 1 18 18 Normal 1 17 17

88 Normal 1 16 16 Normal 1 15 15

89 Normal 1 19 19 Normal 1 18 18

90 Normal 1 16 16 Normal 1 15 15JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 54 of 217

APPENDIX 2 (Continued)

Group : G 3 Dose : 1 00 mg/kg body weight/day

Rat

N° Observation During Treatment Period (Gestation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

76 Normal 19 44 26 Normal 19 43 25

77 Normal 22 47 26 Normal 22 46 25

78 Normal 21 42 22 Normal 21 41 21

79 Normal 17 39 23 Normal 17 38 22

80 Normal 22 47 26 Normal 22 46 25

81 Normal 18 40 23 Normal 18 39 22

82 Normal 18 41 24 Normal 18 40 23

83 Normal 18 43 26 Normal 18 42 25

84 Normal 18 41 24 Normal 18 40 23

85 Normal 19 41 23 Normal 19 40 22

86 Normal 19 41 23 Normal 19 40 22

87 Normal 18 43 26 Normal 18 42 25

88 Normal 16 39 24 Normal 16 38 23

89 Normal 19 41 23 Normal 19 40 22

90 Normal 16 38 23 Normal 16 37 22JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 55 of 217

APPENDIX 2 (Continued)

Group : G 3 Dos e : 1 00 mg/kg body weight/day

Rat

N° Observation During Treatment Period (Lactation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

76

- - - - - - - -

77

- - - - - - - -

78 Normal 42 46 5 Normal 42 45 4

79 Normal 39 43 5 Normal 39 42 4

80

- - - - - - - -

81 Normal 40 44 5 Normal 40 43 4

82 Normal 41 45 5 Normal 41 44 4

83

- - - - - - - -

84 Normal 41 45 5 Normal 41 44 4

85 Normal 41 45 5 Normal 41 44 4

86 Normal 41 45 5 Normal 41 44 4

87

- - - - - - - -

88 Normal 39 43 5 Normal 39 42 4

89 Normal 41 45 5 Normal 41 44 4

90 Normal 38 42 5 Normal 38 41 4

Note: - = Not Applicable

Key: Rat N° 76, 77, 80, 83 and 87 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 56 of 217

APPENDIX 2 (Continued)

Group : G4 Dose : 1000 mg/kg body weight/day

Rat

N° Observation During Treatment Period (Pre

- mating and Mating Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

106 Normal 1 16 16 Normal 1 15 15

107 Normal 1 16 16 Nor mal 1 15 15

108 Normal 1 16 16 Normal 1 15 15

109 Normal 1 18 18 Normal 1 17 17

110 Normal 1 23 23 Normal 1 22 22

111 Normal 1 16 16 Normal 1 15 15

112 Normal 1 19 19 Normal 1 18 18

113 Normal 1 16 16 Normal 1 15 15

114 Normal 1 16 16 Normal 1 15 15

115 Normal 1 19 19 Normal 1 18 18

116 Normal 1 18 18 Normal 1 17 17

117 Normal 1 18 18 Normal 1 17 17

118 Normal 1 22 22 Normal 1 21 21

119 Normal 1 17 17 Normal 1 16 16

120 Normal 1 16 16 Normal 1 15 15JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 57 of 217

APPENDIX 2 (Continued)

Group : G4 Dose : 1000 mg/kg body weight/day

Rat

N° Observation During Treatment Period ( Gestation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

106 Normal 16 41 26 Normal 16 40 25

107 Normal 16 39 24 Nor mal 16 38 23

108 Normal 16 38 23 Normal 16 37 22

109 Normal 18 40 23 Normal 18 39 22

110 Normal 23 48 26 Normal 23 47 25

111 Normal 16 38 23 Normal 16 37 22

112 Normal 19 41 23 Normal 19 40 22

113 Normal 16 41 26 Normal 16 40 25

114 Normal 16 41 26 Normal 16 40 25

115 Normal 19 42 24 Normal 19 41 23

116 Normal 18 41 24 Normal 18 40 23

117 Normal 18 41 24 Normal 18 40 23

118 Normal 22 44 23 Normal 22 43 22

119 Normal 17 39 23 Normal 17 38 22

120 Normal 16 41 26 Normal 16 40 25JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 58 of 217

APPENDIX 2 (Con tinued)

Group : G4 Dose : 1000 mg/kg body weight/day

Rat

N° Observation During Treatment Period ( L actation Period)

Morning

Observation First

Day Last

Day Total

Days Post Dosing

Observation First

Day Last

Day Total

Days

106

- - - - - - - -

107 Normal 39 43 5 Normal 39 42 4

108 Normal 38 42 5 Normal 38 41 4

109 Normal 40 44 5 Normal 40 43 4

110

- - - - - - - -

111 Normal 38 42 5 Normal 38 41 4

112 Normal 41 45 5 Normal 41 44 4

113

- - - - - - - -

114

- - - - - - - -

115 Normal 42 46 5 Normal 42 45 4

116 Normal 41 45 5 Normal 41 44 4

117 Normal 41 45 5 Normal 41 44 4

118 Normal 44 48 5 Normal 44 47 4

119 Normal 39 43 5 Normal 39 42 4

120

- - - - - - - -

Note: - = Not Applicable

Key: Rat N° 106, 110, 113, 114 and 120 were non

- pregnantJRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 59 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 3

Weekly Body Weight (g) of Individual Male Rat (P)

Group: G1 Dose: 0 mg/kg body weight/day

Rat N° Treatme nt Days

1 8 15 22 29 36 43

1 304.5 326.7 356.4 368.5 396.2 423.8 429.6

2 352.9 371 .0 406.6 428.3 458 .0 481 .0 504.1

3 284.7 304.1 329.6 345.1 368.1 386.1 401 .0

4 296.4 318.3 349.1 361 .0 376.8 393.2 400.4

5 301.7 331.9 354.9 367.9 389.8 417.7 429.8

6 276 .0 296.7 315.7 330.8 346.3 353 .0 363.9

7 311.9 335.3 350.8 369.7 374.3 389.3 391.3

8 319.7 338.5 358.5 384.1 398.3 416.8 426.2

9 316.1 339.2 363 .0 385.2 399 .0 409.8 415.9

10 324.2 345.1 367.1 379.3 396.2 412.2 414.5

11 334.1 360 .0 383.8 406.7 420. 6 436.6 445.7

12 340 .0 370.5 406.3 436 .0 459.7 488.9 507.5

13 334.6 360.8 382.8 401.3 416.8 441.1 461.8

14 337.7 370.5 403.3 420.2 447 .0 472.8 489.9

15 301.8 336.1 369.3 396.1 427.6 455.7 479.8JRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 60 of 217

APPENDIX 3 (Continued)

Group: G 2 Dose: 1 0 mg/kg body w eight/day

Rat N° Treatment Days

1 8 15 22 29 36 43

31 316.1 338.3 355.2 366 .0 381.2 390.9 398.5

32 292.9 308.9 329 .0 340.8 354.2 367.2 378.4

33 294.7 328.4 355.8 376.1 395.5 415.1 424 .0

34 299.3 324.1 349.3 370.3 382.8 396.4 408.3

35 307.9 338.4 360 .4 381.9 400.6 414 .0 429.5

36 312.6 329.5 346.4 365.3 378.6 388.1 402.7

37 321.7 350.1 370.4 380.7 400.1 419.4 431.4

38 329 .0 351.3 372.1 390.1 404.8 416.3 427.6

39 326.7 344.4 356.3 376.5 388.8 400.3 415 .0

40 334.1 360 .0 375.1 380.4 393.9 410.6 423.2

41 335 .0 355.3 380.3 387.6 403.1 426.2 443.8

42 290.7 312 .0 334.3 341.8 350.5 361.7 371.2

43 343.8 368.9 392.5 405.1 424.8 439.4 449.2

44 276.3 297.1 316.8 331.8 350.8 370.7 386.4

45 339.8 368.4 389.3 402.6 412.6 426.6 438.1JRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 61 of 217

APPENDIX 3 (Continued)

Group: G 3 Dose: 10 0 mg/kg body weight/day

Rat N° Treatment Days

1 8 15 22 29 36 43

61 306.1 327.1 341.2 358.3 365.8 375.3 390.2

62 345.4 360.4 385.3 392.4 401.9 420 .0 439.2

63 287.5 312.3 330.1 334.3 344.8 355.5 370 .0

64 297.1 324.1 340.9 355.2 371 .2 389.4 412.4

65 278 .0 294 .0 310.8 319.9 328 .0 344.4 356.6

66 310.8 338.1 355.7 371.5 382.6 394.7 413 .0

67 315.5 355.1 385.2 398.6 425.2 448.3 469.7

68 313.4 341.7 357.9 364.9 377.8 389.9 400.4

69 318.8 343.8 362.8 379 .0 402.6 416.7 439.2

70 325.6 3 56.1 380.5 391.7 407.5 423.8 438.7

71 330 .0 353.2 364.9 376.9 388.5 398.6 414.4

72 331.1 368.1 391.2 402.2 427.1 446.2 458.6

73 333 .0 356 .0 376.8 388.8 404 .0 416.8 422.4

74 336.5 353.7 366.2 374.4 377.1 382.2 394.1

75 294.7 309 .0 323.6 330.8 338.1 351 .4 362.4JRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 62 of 217

APPENDIX 3 (Continued)

Group: G 4 Dose: 1000 mg/kg body weight/day

Rat N° Treatment Days

1 8 15 22 29 36 43

91 319 .0 340.2 353.1 375.8 386.9 400.6 406.7

92 286.4 304.6 322.4 333.6 353.8 364.9 378.4

93 289.1 313 .0 336 .0 352.1 371.6 385.2 39 4 .0

94 296.5 326.2 345.8 362.5 376.1 394 .0 408.4

95 304 .0 332.4 353.4 370 .0 388.9 394.7 405.3

96 309.3 335.9 350.7 363.3 379.5 386.8 395.3

97 314.4 345 .0 375.7 387 .0 405.8 431.1 445.3

98 317.8 334.9 356.6 371.4 385.4 397 .0 407.4

99 325.7 354.2 380.1 385.1 416.9 426 .0 432.7

100 326.6 355 .0 363.2 376.1 383.7 399.6 410 .0

101 333.4 358.8 377.8 385.8 409 .0 422.7 431.9

102 336.7 368.9 382.9 396.9 416.1 432.1 453.2

103 340.4 374.1 395.3 414.2 433.7 450.8 466.1

104 278.3 301.8 325.3 343.1 355 .0 372.3 383 .6

105 328.1 365 .0 388.5 403 .0 419.8 436.7 452.2JRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 63 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 4

Weekly Body Weight (g) of Individual Female Rat (P)

Group: G1 Dose: 0 mg/kg body weight/day

Rat

N° Pre

- mating and Mating Day Gestation Day Lactation Day

1 8 15 22 0 7 14 20 25 0 4

16 208.1 219 .0 224.6

- 243.3 264.3 294.1 360.6

- 267.5 257.8

17 240.7 258.2 269.5

- 273.9 291.4 318.3 379.8

- 289.2 288.4

18 202.2 217.2 238.6

- 239.7 255.1 280.6 348.7

- 235.4 249.4

19 204.7 218.4 229.6

- 236.3 266.6 302.5 365.8

- 272.6 276.2

20 206.3 217.5 227.9

- 226.6 253.8 275.6 312.8

- 284.9 286.9

21 193.7 204 .0 210 .0

- 219.3 247.1 263.1 293.8 291.4

- -

22 213.9 224 .9 232 .0

- 235.9 264.1 293.4 346.7

- 271.9 269.8

23 215.9 224.4 235.6

- 237.9 267.8 298.8 366.6

- 265.5 287.1

24 217 .0 226.3 236.9

- 234.2 260.1 290.9 352.9

- 252 .0 269.7

25 225.7 233.1 240.7

- 232.7 262.2 273.1 291.2 290.9

- -

26 227.8 241.6 251.7 - 2 65.2 281.8 315.5 308.7 311.7

- -

27 228 .0 241.8 248.4

- 249.9 273.9 297.5 337.6

- 305.1 301.4

28 229.3 237.8 246 .0

- 247.4 272.2 279.5 284 .0 280.9

- -

29 238.1 244.9 250.6

- 261.9 305.1 340.6 376.8

- 356.5 348.7

30 203.4 209.8 219.6

- 227.6 245.1 269.7 317.7

- 244.3 247.3

Key:

- = Not applicable

Note: Rat N° 21, 25, 26 and 28 w ere non

- pregnantJRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 64 of 217

APPENDIX 4 (Continued)

Group: G 2 Dose: 10 mg/kg body weight/day

Rat

N° Pre

- mating and Mating Day Gestation Day Lactation Day

1 8 15 22 0 7 14 20 25 0 4

46 217.4 228 .0 239.6

- 232.6 262.8 289.2 346.9

- 269.8 272.8

47 205.3 213.9 221.5

- 230.2 250 .0 280.2 333.8

- 246.8 249.9

48 204.8 212.4 222.8

- 227.1 255.1 283 .0 342.3

- 242.9 259 .0

49 204.2 213.4 224 .0

- 230.2 254.9 288.6 345.8

- 284.9 288.4

50 208.4 220.3 231.2

- 233 .0 266.8 304.2 377.8

- 267.5 271.4

51 217.5 228.4 237.4

- 237 .0 257.7 280.1 325.8

- 278.6 280.6

52 219.4 234.1 241.7

- 251.8 277.1 308.8 377 .0

- 269.8 274 .0

53 224.1 218.9 239.6

- 236.7 258.2 286.8 349.2

- 246.6 268.2

54 225.4 239.7 2 47.9

- 252.8 268.3 300.1 375.8

- 276.5 284.7

55 226.9 235.7 239.4

- 249.5 274.3 311.8 368.2

- 264.3 277.5

56 229.4 237 .0 249.5

- 244.3 273 .0 279.8 286.1 294.5

- -

57 204.1 217.5 230.9

- 245.4 260.8 290.6 341.3

- 265.9 269.7

58 241 .0 250.7 261.5

- 263.7 296.8 326.9 393 .0

- 297 .0 308.7

59 189.4 203.1 214.5

- 214.6 238.4 261.4 319.7

- 243.6 244.2

60 235.1 253.4 270.9

- 276.8 296.9 321.3 369.8

- 283 .0 290.6

Key:

- = Not applicable

Note: Rat N ° 56 w as non

- pregnantJRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 65 of 217

APPENDIX 4 (Continued)

Group: G 3 Dose: 10 0 mg/kg body weight/day

Rat

N° Pre

- mating and Mating Day Gestation Day Lactation Day

1 8 15 22 0 7 14 20 25 0 4

76 206 .0 218.9 224 .0

- 229.2 262.3 273.4 272.8 269 .0

- -

77 250.1 266.5 270.4

- 301.5 305.1 308.7 316.5 318.9

- -

78 197.4 210.5 217. 3 - 230 .0 258.8 296 .0 342.3

- 238 .0 254 .0

79 199.8 214.3 224.5

- 220.7 246.9 273.6 335.2

- 242.2 248.7

80 202.4 212.3 221.1

- 234.6 251.7 264.9 245.8 249.5

- -

81 208.9 216 .0 224.9

- 227.3 254.3 280.4 344.9

- 265.9 277.4

82 214.1 225.4 232.1

- 238.4 25 6.8 280.5 321.2

- 289.5 289 .0

83 213.5 224.7 236.8

- 242.3 263.8 271 .0 277.1 279.1

- -

84 215.9 227.9 232.7

- 236.3 265.1 304 .0 381.8

- 275.4 276.2

85 223.5 231.8 237.8

- 251.4 274.9 304.5 360.8

- 280.6 299 .0

86 225.4 240.1 253.6

- 258.5 290.8 326.8 39 0.6

- 282.2 298 .0

87 225 .0 235.6 237.6

- 236.9 256.7 272.4 281.8 279.8

- -

88 233.3 245.7 248.1

- 247.2 274.9 300 .0 339.2

- 305 .0 294 .0

89 239.7 252.8 261.8

- 276.5 289 .0 314.4 367.1

- 284 .0 285.5

90 204.7 213.4 224.8

- 229.9 248.9 275 .0 327.8

- 250.1 254.7

Key:

- = Not applicable

Note: Rat N° 76, 77, 80, 83 and 87 w ere non

- pregnantJRF Stu dy Number: 496 -1- 04

- 4306 ( Final Report) Page 66 of 217

APPENDIX 4 (Continued)

Group: G 4 Dose: 1000 mg/kg body weight/day

Rat

N° Pre

- mating and Mating Day Gestation Day Lactation Day

1 8 15 22 0 7 14 20 25 0 4

106 219 .1 231 .0 239.7

- 239.1 269.1 281.1 294.7 291.3

- -

107 201.3 208.7 219.6

- 225.8 257.4 285.7 347.1

- 270.2 276.3

108 201.5 210.4 224.5

- 224.1 235.1 272.9 339.9

- 260.8 272.5

109 204.7 216.4 219.4

- 224.3 240.3 268.7 336.9

- 258.2 258.3

110 209.9 220.3 230.4 248.3 250.1 261.1 270.9 274.6 275.8

- -

111 208.7 216.7 225.5

- 225.4 245.8 267.3 318.8

- 271 .0 265.8

112 210.4 218.9 230.8

- 229.8 259.7 292.8 361.2

- 267.8 273.7

113 222.4 234.8 250.9

- 249.6 275 .0 282.8 296.1 294.8

- -

114 221.4 235.1 256.5

- 265.9 276 .0 279.1 276.1 277.8

- -

115 222.8 236 .0 243 .0

- 249 .0 274.3 302.3 375.6

- 277.8 299.9

116 229.1 236.4 242.6

- 242.7 268.4 302.6 380.5

- 266.8 285.3

117 230.1 237.4 246.7

- 250.2 271.3 303 .0 377.8

- 280.8 287.4

118 237.8 255.6 262.1

- 292.1 3 11.1 317.2 375.9

- 302.6 305.8

119 194.7 203.4 218.6

- 221.9 247 .0 281.8 356.8

- 266.5 279.5

120 239.7 247.3 260.4

- 264.9 300.1 306.7 319.2 312.3

- -

Key:

- = Not applicable

Note: Rat N° 106, 110, 113, 114 and 120 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 67 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 5

Weekly Body Weight Change (%) of Individual Male Rat (P)

Group : G1 Dose : 0 mg/kg body weight/day

Rat N ° During Treatment Days

1-8 8- 15 15

- 22 22

- 29 29

- 36 36

- 43 1- 43

1 7.29 9.09 3.40 7.52 6.97 1.37 41.08

2 5.13 9.60 5.34 6.93 5.02 4.80 42.84

3 6.81 8.39 4.70 6.66 4.89 3.86 40.85

4 7.39 9.68 3.41 4.38 4.35 1.83 35.09

5 10.01 6.93 3.66 5.95 7.16 2.90 42 .46

6 7.50 6.40 4.78 4.69 1.93 3.09 31.85

7 7.50 4.62 5.39 1.24 4.01 0.51 25.46

8 5.88 5.91 7.14 3.70 4.64 2.26 33.31

9 7.31 7.02 6.12 3.58 2.71 1.49 31.57

10 6.45 6.37 3.32 4.46 4.04 0.56 27.85

11 7.75 6.61 5.97 3.42 3.80 2.08 33.40

12 8.97 9.66 7.31 5.44 6.35 3.80 49.26

13 7.83 6.10 4.83 3.86 5.83 4.69 38.02

14 9.71 8.85 4.19 6.38 5.77 3.62 45.07

15 11.37 9.88 7.26 7.95 6.57 5.29 58.98JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 68 of 217

APPENDIX 5 (Continued)

Group : G2 Dose : 10 mg/kg body weight/day

Rat N° During Treatment Days

1-8 8- 15 15- 23 22

- 29 29

- 36 36

- 43 1- 43

31 7.02 5.00 3.04 4.15 2.54 1.94 26.07

32 5.46 6.51 3.59 3.93 3.67 3.05 29.19

33 11.44 8.34 5.71 5.16 4.96 2.14 43.88

34 8.29 7.78 6.01 3.38 3.55 3.00 36.42

35 9.91 6.50 5.97 4.90 3.34 3.74 39.49

36 5.41 5.13 5.46 3.64 2.5 1 3.76 28.82

37 8.83 5.80 2.78 5.10 4.82 2.86 34.10

38 6.78 5.92 4.84 3.77 2.84 2.71 29.97

39 5.42 3.46 5.67 3.27 2.96 3.67 27.03

40 7.75 4.19 1.41 3.55 4.24 3.07 26.67

41 6.06 7.04 1.92 4.00 5.73 4.13 32.48

42 7.33 7.15 2.24 2.55 3.20 2.63 27.69

43 7.30 6.40 3.21 4.86 3.44 2.23 30.66

44 7.53 6.63 4.73 5.73 5.67 4.24 39.85

45 8.42 5.67 3.42 2.48 3.39 2.70 28.93JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 69 of 217

APPENDIX 5 (Continued)

Group : G 3 Dose : 1 00 mg/kg body weight/day

Rat N° During Treatment Days

1-8 8- 15 15

- 22 22

- 29 29

- 36 36

- 43 1- 43

61 6.86 4.31 5.01 2.09 2.60 3.97 27.47

62 4.34 6.91 1.84 2.42 4.50 4.57 27.16

63 8.63 5.70 1.27 3.14 3.10 4.08 28.70

64 9.09 5.18 4.19 4.50 4.90 5.91 38.81

65 5.76 5.71 2.93 2.53 5.00 3.54 28.27

66 8.78 5.21 4.44 2.99 3.16 4.64 32.88

67 12.55 8.48 3 .48 6.67 5.43 4.77 48.87

68 9.03 4.74 1.96 3.54 3.20 2.69 27.76

69 7.84 5.53 4.47 6.23 3.50 5.40 37.77

70 9.37 6.85 2.94 4.03 4.00 3.52 34.74

71 7.03 3.31 3.29 3.08 2.60 3.96 25.58

72 11.17 6.28 2.81 6.19 4.47 2.78 38.51

73 6.91 5.84 3.18 3.91 3.17 1 .34 26.85

74 5.11 3.53 2.24 0.72 1.35 3.11 17.12

75 4.85 4.72 2.22 2.21 3.93 3.13 22.97JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 70 of 217

APPENDIX 5 (Continued)

Group : G4 Dose : 1000 mg/kg body weight/day

Rat N° During Treatment Days

1-8 8- 15 15

- 22 22

- 29 29

- 36 36

- 43 1- 43

91 6.65 3.79 6.43 2.95 3.54 1.52 27.49

92 6.35 5.84 3.47 6.06 3.14 3.70 32.12

93 8.27 7.35 4.79 5.54 3.66 2.28 36.29

94 10.02 6.01 4.83 3.75 4.76 3.65 37.74

95 9.34 6.32 4.70 5.11 1.49 2.69 33.32

96 8.60 4.41 3.59 4.46 1.92 2.20 27.80

97 9.73 8.90 3.01 4.86 6.23 3.29 41.63

98 5.38 6.48 4.15 3.77 3.01 2.62 28.19

99 8.75 7.31 1.32 8.26 2.18 1.57 32.85

100 8.70 2.31 3.55 2.02 4.14 2.60 25.54

101 7.62 5.30 2.12 6.01 3.35 2.18 29.54

102 9.56 3.80 3.66 4.84 3.85 4.88 34.60

103 9.90 5.67 4.78 4.71 3.94 3.39 36.93

104 8.44 7 .79 5.47 3.47 4.87 3.04 37.84

105 11.25 6.44 3.73 4.17 4.03 3.55 37.82JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 71 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 6

Weekly Body Weight Change (%) of Individual Female Rat (P)

Group : G1 Dose : 0 mg/kg body weight/day

Rat

N° Pre

- mating Period

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 1- 15 0-7 7- 14 14

- 20 0- 20 0-4

16 5.24 2.56 7.93 8.63 11.28 22.61 48.21

- 3.63

17 7.27 4.38 11.97 6.39 9.23 19.32 38.66

- 0.28

18 7.42 9.85 18.00 6.42 10.00 24.27 45.47 5.95

19 6.69 5.13 12.16 12.82 13.47 20.93 54.80 1.32

20 5.43 4.78 10.47 12.00 8.59 13.50 38.04 0.70

21 5.32 2.94 8.42 12.68 6.48 11.67 33.97

-

22 5.14 3.16 8.46 11.95 11.09 1 8.17 46.97

- 0.77

23 3.94 4.99 9.12 12.57 11.58 22.69 54.10 8.14

24 4.29 4.68 9.17 11.06 11.84 21.31 50.68 7.02

25 3.28 3.26 6.65 12.68 4.16 6.63 25.14

-

26 6.06 4.18 10.49 6.26 11.96

- 2.16 16.40

-

27 6.05 2.73 8.95 9.60 8.62 13.48 35.09

- 1.21

28 3.71 3.45 7.28 10.02 2.68 1.61 14.79

-

29 2.86 2.33 5.25 16.49 11.64 10.63 43.87

- 2.19

30 3.15 4.67 7.96 7.69 10.04 17.80 39.59 1.23

Key:

- = Not applicable

Note: Rat N° 21, 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 72 of 217

APPENDIX 6 (Continued)

Group : G2 Dose : 10 mg/kg body weight/day

Rat

N° Pre

- mating Period

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 1- 15 0-7 7- 14 14

- 20 0- 20 0-4

46 4.88 5.09 10.21 12.98 10.05 19.95 49.14 1.11

47 4.19 3.55 7.89 8.60 12.08 19.13 45.00 1.26

48 3.71 4.90 8.79 12.33 10.94 20.95 50.73 6.63

49 4.51 4.97 9.70 10.73 13.22 19.82 50.22 1.23

50 5.71 4.95 10.94 14.51 14.02 24.19 62.15 1.46

51 5.01 3.94 9.15 8.73 8.69 16.32 37.47 0.72

52 6.70 3.25 10.16 10.05 11.44 22.09 49.72 1.56

53

- 2.32 9.46 6.92 9.08 11.08 21.7 6 47.53 8.76

54 6.34 3.42 9.98 6.13 11.85 25.22 48.66 2.97

55 3.88 1.57 5.51 9.94 13.67 18.09 47.58 4.99

56 3.31 5.27 8.76 11.75 2.49 2.25 17.11

-

57 6.57 6.16 13.13 6.28 11.43 17.45 39.08 1.43

58 4.02 4.31 8.51 12.55 10.14 20.22 49.03 3.94

59 7.23 5 .61 13.25 11.09 9.65 22.30 48.97 0.25

60 7.78 6.91 15.23 7.26 8.22 15.09 33.60 2.69

Key:

- = Not applicable

Note: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 73 of 217

APPENDIX 6 (Continued)

Group : G 3 Dose : 1 00 mg/kg body weight/day

Rat

N° Pre

- mating Period

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 1- 15 0-7 7- 14 14

- 20 0- 20 0-4

76 6.26 2.33 8.74 14.44 4.23

- 0.22 19.02

-

77 6.56 1.46 8.12 1.19 1.18 2.53 4.98

-

78 6.64 3.23 10.08 12.52 14.37 15.64 48.83 6.72

79 7.26 4.76 12.36 11.87 10.81 22.51 51.88 2.68

80 4.89 4.15 9.24 7.29 5.24

- 7.21 4.77

-

81 3.40 4.12 7.66 11.88 10.26 23.00 51.74 4.32

82 5.28 2.97 8.41 7.72 9.23 14.51 34.73

- 0.17

83 5.25 5.38 10.91 8.87 2.73 2.25 14.36

-

84 5.56 2.11 7.78 12.19 14.67 25.59 61.57 0.29

85 3.71 2.59 6.40 9.3 5 10.77 18.49 43.52 6.56

86 6.52 5.62 12.51 12.50 12.38 19.52 51.10 5.60

87 4.71 0.85 5.60 8.36 6.12 3.45 18.95

-

88 5.32 0.98 6.34 11.21 9.13 13.07 37.22

- 3.61

89 5.47 3.56 9.22 4.52 8.79 16.76 32.77 0.53

90 4.25 5.34 9.82 8.26 10.49 19.20 42.58 1.84

Key:

- = Not applicable

Note: Rat N° 76, 77, 80, 83 and 87 w ere non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 74 of 217

APPENDIX 6 (Continued)

Group : G4 Dose : 1000 mg/kg body weight/day

Rat

N° Pre

- mating Period

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 1- 15 0-7 7- 14 14

- 20 0- 20 0-4

106 5.43 3.77 9.40 12.55 4.46 4.84 23.25

-

107 3.68 5.22 9.09 13.99 10.99 21.49 53.72 2.26

108 4.42 6.70 11.41 4.91 16.08 24.55 51.67 4.49

109 5.72 1.39 7.18 7.13 11.82 25.38 50.20 0.04

110 4.95 4.58 9.77 4.40 3.75 1.37 9.80

-

111 3.83 4.06 8.05 9.05 8.75 19.27 41.44

- 1.92

112 4.04 5.44 9.70 13.01 12.75 23.36 57.18 2.20

113 5.58 6.86 12.81 10.18 2.84 4.70 18.63

-

114 6.19 9.10 15.85 3.80 1.12

- 1.07 3.84

-

115 5.92 2.97 9.07 10.16 10.21 24.25 50.84 7.96

116 3.19 2.62 5.89 10.59 12.7 4 25.74 56.78 6.93

117 3.17 3.92 7.21 8.43 11.68 24.69 51.00 2.35

118 7.49 2.54 10.22 6.50 1.96 18.51 28.69 1.06

119 4.47 7.47 12.28 11.31 14.09 26.61 60.79 4.88

120 3.17 5.30 8.64 13.29 2.20 4.08 20.50

-

Key:

- = Not applicable

Note: Rat N° 106, 110, 113, 114 and 120 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 75 of 217

Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127

- 68

- 4; US EPA HPV 3) in Wistar Rats

APPENDIX 7

F oo d Consumption (g ) of Individual Male Rat (P)

Group: G1 Dose: 0 mg/kg body weight/day

Rat N° During Tr eatment Days

1-8 8- 15 23- 29 29- 36 36

- 43 43

- TS

1 25.0 26.0 27.4 24.5 26.4 28.3

2 27.3 30.8 27.9 28.2 28.7 27.5

3 22.6 22.1 23.2 23.6 23.1 23.5

4 22.9 23.9 23.0 22.9 23.2 23.9

5 25.3 25.0 25.4 26.1 25.7 26.0

6 24.3 24.1 23.4 22.4 21.8 23.2

7 24.7 23 .4 22.5 21.7 21.9 22.2

8 23.8 22.5 22.7 22.2 22.2 22.7

9 24.7 23.5 23.6 21.3 20.5 21.0

10 26.0 24.0 23.1 21.9 21.0 20.1

11 25.0 26.3 24.9 25.3 23.8 24.8

12 26.6 30.1 29.3 28.0 27.3 30.0

13 25.1 27.0 26.2 25.4 25.5 27.3

14 27.7 29.8 29.8 29.9 29.3 31 .8

15 27.0 28.7 31.3 30.1 30.0 36.3

Key: TS = Terminal SacrificeJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 76 of 217

APPENDIX 7 (Continued)

Group: G2 Dose: 10 mg/kg body weight/day

Rat N° During Treatment Days

1-8 8- 15 23

- 29 29

- 36 36

- 43 43

- TS

31 24.8 23.2 23.1 21.5 20.6 22.4

32 21.6 22.8 21.5 20.6 2 0.7 21.7

33 25.8 25.1 24.4 24.7 22.9 26.0

34 25.4 23.4 24.4 23.6 22.9 24.3

35 25.1 24.8 24.1 22.5 23.1 23.9

36 23.7 23.1 23.0 22.7 24.2 25.5

37 26.9 26.4 25.0 24.9 24.9 27.4

38 23.9 23.4 23.8 22.2 21.6 22.7

39 26.0 24.2 24.6 26.2 27.0 27.1

40 27.3 25.1 22.6 24.6 23.8 25.5

41 27.3 28.9 26.5 27.2 27.2 30.5

42 23.2 22.8 21.7 19.3 19.8 20.4

43 25.0 25.7 25.4 23.3 23.1 23.8

44 21.9 21.8 22.7 23.8 23.4 23.9

45 27.2 28.3 24.7 24.3 23.6 24.1

Key: TS = Terminal SacrificeJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 77 of 217

APPENDIX 7 (Continued)

Group : G3 Dose: 1 00 mg/kg body weight/day

Rat N° During Treatment Days

1-8 8- 15 23

- 29 29

- 36 36

- 43 43

- TS

61 23.3 22.8 23.3 21.8 22.3 22.9

62 26.0 25.2 23.4 22.1 23.5 22.8

63 24.6 24.0 21.7 21.5 21.0 22.6

64 25.8 24.6 23.6 23.1 24.4 24.3

65 24.1 22.5 21.5 21.7 22.2 21.8

66 25.8 24.8 22.7 22.0 23.2 25.1

67 27.0 28.4 27.4 27.6 28.0 28.4

68 24.6 23.5 22.7 21.5 20.7 21.6

69 24.2 23.4 24.0 23.6 22.9 23.7

70 26.7 26.8 25.1 23.0 24.6 25.6

71 23.9 22.2 25.0 20.6 20.2 22.3

72 26.0 27.5 32.5 27.0 30.0 30.1

73 26.4 26.8 26.1 23.8 23.7 24.6

74 26.6 24.2 25.1 19.1 20.4 23.0

75 24.4 21.6 21.7 19.1 19.8 21.9

Key: TS = Terminal SacrificeJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 78 of 217

APPENDIX 7 (Continued)

Group: G4 Dose: 1000 mg/kg body weight/day

Rat N° During Treatment Days

1-8 8- 15 23- 29 29

- 36 36

- 43 43

- TS

91 22.5 20.5 20.6 21.5 20.2 21.9

92 21.0 20.9 21.5 19.6 19.5 19.3

93 23.5 24.1 24.5 22.8 21.8 22.6

94 24.0 24.7 24.9 24.2 23.7 26.1

95 24.6 23.5 24.8 22.9 21.3 23.8

96 24.1 23.6 22.5 22.3 22.9 23.3

97 26.7 28.6 25.8 26.4 26.1 28.5

98 23.7 25. 2 26.5 24.3 25.1 26.1

99 27.5 31.3 29.5 26.7 24.1 27.0

100 26.5 25.7 22.5 24.2 23.0 26.0

101 27.0 26.6 25.7 26.0 24.5 25.4

102 27.1 27.1 25.0 25.8 27.1 27.6

103 25.5 26.6 25.6 26.0 25.3 25.9

104 20.7 23.1 22.1 22.2 21.6 22.5

105 27.3 27.6 27.2 27.9 28.3 27.5

Key: TS = Terminal SacrificeJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 79 of 217

Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127

- 68

- 4; US EPA HPV 3) in Wistar Rats

APPENDIX 8

F oo d Consumption (g ) of Individual Female Rat (P)

Group: G1 Do se: 0 mg/kg body weight/day

Rat N° Pre

- mating Period

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 0-7 7- 14 14

- 20 0-4

16 17.3 18.5 21.1 22.5 27.7 26.7

17 23.0 24.1 23.0 23.5 22.2 22.3

18 21.6 19.7 18.5 18.8 19.9 23.7

19 20.1 21.8 23.7 24.7 22.4 23.9

20 17.8 18.0 19.6 21.0 23.4 20.6

21 17.2 17.4 20.7 18.2 19.8

-

22 17.1 17.3 20.3 21.2 20.9 23.0

23 17.2 16.6 19.7 21.2 23.1 27.4

24 19.5 18.5 21.3 22.1 22.0 28.5

25 20.4 21.2 20.5 19.5 19.1

-

26 23.1 22.5 23.7 26.1 18.5

-

27 18 .8 18.6 19.7 21.0 21.9 22.5

28 19.1 19.8 22.2 19.9 18.8

-

29 19.6 20.0 23.8 24.5 31.3 21.0

30 16.9 18.0 19.5 18.8 20.2 18.7

Key:

- = Not applicable

Note: Rat N° 21, 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 80 of 217

APPENDIX 8 (Continued)

Group: G2 Dose: 10 mg/kg bod y weight/day

Rat N° Pre

- mating Period

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 0-7 7- 14 14

- 20 0-4

46 19.6 19.4 20.3 22.3 24.8 27.8

47 18.6 19.0 20.5 19.7 19.9 21.6

48 15.6 16.9 21.1 21.0 21.0 24.1

49 17.4 18.4 20.6 22.4 28.5 18.2

50 18.0 18.9 23.7 24.8 27.8 26.2

51 18.7 18.8 20.6 21.2 22.9 20.1

52 19.5 20.3 22.6 23.8 22.9 31.7

53 18.6 19.8 19.9 22.8 21.2 27.5

54 20.1 18.8 19.3 21.2 22.3 25.4

55 20.0 19.7 22.1 23.5 21.8 24.2

56 18.3 18.0 19.6 21.6 20.3

-

57 18.2 18.5 2 0.2 20.8 22.6 16.6

58 20.1 19.5 23.2 23.0 25.4 26.9

59 17.6 16.9 19.6 20.7 22.9 22.5

60 19.2 20.1 23.1 22.4 24.3 24.2

Key:

- = Not applicable

Note: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 81 of 217

APPENDIX 8 (Continued)

Group: G3 Dose: 1 00 mg/kg body weight/day

Rat N° P re

- mating Period

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 0-7 7- 14 14

- 20 0-4

76 19.4 17.1 23.8 22.2 19.2

-

77 21.6 21.9 20.7 19.5 18.5

-

78 19.0 18.5 19.0 20.9 26.4 19.7

79 17.7 18.3 18.7 21.9 21.2 26.6

80 16.6 16.9 19.4 16 .1 13.4

-

81 18.1 17.8 21.3 23.0 25.6 26.9

82 19.8 19.3 19.1 21.3 21.0 19.2

83 16.7 17.0 18.6 20.4 19.3

-

84 17.7 17.1 19.1 23.0 23.9 31.7

85 18.0 18.8 21.0 22.1 23.0 23.7

86 20.0 21.3 24.1 24.6 25.3 25.0

87 17.7 16.1 18.3 18.5 17.4

-

88 20.0 17.2 21.0 22.7 22.0 18.8

89 19.8 20.2 19.6 20.5 22.6 19.3

90 17.5 18.8 19.9 21.8 21.2 23.2

Key:

- = Not applicable

Note: Rat N° 76, 77, 80 83 and 87 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 82 of 217

APPENDIX 8 (Continued)

Group: G4 Dose: 1000 mg/kg body weight/day

Rat N° Pre

- mating Per iod

(Days) Gestation Period

(Days) Lactation Period

(Days)

1-8 8- 15 0-7 7- 14 14

- 20 0-4

106 19.2 19.2 20.2 21.3 21.8

-

107 17.0 17.5 20.5 22.8 23.2 23.5

108 17.4 17.6 17.7 20.8 22.2 22.8

109 16.8 17.0 18.0 18.5 20.6 22.3

110 18.6 18.4 18.4 17.0 27 .4

-

111 17.6 18.5 18.6 20.2 21.6 21.2

112 16.9 17.8 20.0 21.7 21.3 23.8

113 19.1 20.1 20.7 21.7 21.8

-

114 17.8 20.8 19.8 18.1 16.3

-

115 19.3 19.6 20.9 22.4 24.2 31.7

116 16.7 17.5 19.8 23.0 24.5 32.2

117 17.9 19.8 19.6 23.1 25.5 31.4

118 20.1 19 .9 23.2 19.3 23.7 26.6

119 16.7 13.1 19.4 23.6 24.9 29.5

120 19.0 19.1 21.0 16.2 20.1

-

Key:

- = Not applicable

Note: Rat N° 106, 110, 113, 114 and 120 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 83 of 217

Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127

- 68

- 4; US EPA HPV 3) in Wistar Rats

APPENDIX 9

Pre

- coital Interval and Duration of Gestation Days of Individual Female Rat (P)

Group: G1 Dose: 0 mg/kg body weight/day

Rat N° Pre

- coital Interval Day Duration of Gestation Day

16 4 23

17 2 22

18 3 23

19 3 22

20 1 23

21 1

-

22 3 22

23 2 22

24 2 22

25 1

-

26 7

-

27 3 23

28 3

-

29 4 23

30 4 22

Key:

- = Not applicable

Note: Rat N° 21, 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 84 of 217

APPENDIX 9 (Continued)

Group: G2 Dose: 10 mg/kg body weight/day

Rat N° Pre

- coital Interval Day Duration of Gestation Day

46 1 23

47 4 22

48 1 23

49 4 22

50 3 23

51 1 23

52 4 22

53 1 22

54 2 22

55 4 22

56 2

-

57 4 22

58 2 22

59 2 22

60 2 23

Key:

- = Not applicable

Note: Rat N° 56 was non

- preg nantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 85 of 217

APPENDIX 9 (Continued)

Group: G3 Dose: 1 00 mg/kg body weight/day

Rat N° Pre

- coital Interval Day Duration of Gestation Day

76 4

-

77 7

-

78 6 21

79 2 22

80 7

-

81 3 22

82 3 23

83 3

-

84 3 23

85 4 22

86 4 22

87 3

-

88 1 23

89 4 22

90 1 22

Key:

- = Not applicable

Note: Rat N° 76, 77, 80, 83 and 87 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 86 of 217

APPENDIX 9 (Continued)

Group: G4 Dose: 1000 mg/kg body weight/day

Rat N° Pre

- coital Interval Day Duration of Gestation Day

106 1

-

107 1 23

108 1 22

109 3 22

110 8

-

111 1 22

112 4 22

113 1

-

114 1

-

115 4 23

116 3 23

117 3 23

118 7 22

119 2 22

120 1

-

Key:

- = Not applicable

Note: Rat N° 106, 110, 113, 114 and 120 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 87 of 21 7



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 10

Clinical Symptoms of Individual Pup s

Group: G1 Dose: 0 mg/kg body weight/day

Rat N° Pup N° Sex Lactatio n Day

0 1 2 3 4

16 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

17 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 F 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

18 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 2

- - - -

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicable, 1 = Normal, 2 = DeadJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 88 of 21 7

APPENDIX 10 (C ontinued)

Group : G1 D ose : 0 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

19 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 1 1 1 1 1

9 M 1 1 1 1 1

10 M 1 1 1 1 1

11 M 1 1 1 1 1

12 M 1 1 1 1 1

13 M 2

- - - -

14 F 1 1 1 1 1

20 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 F 1 1 1 1 1

22 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 2

- -

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

23 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 2

- - - -

Key: M = Male, F = Female,

- = Not applicable, 1 = Normal, 2 = Dead

Note: Rat N° 21 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 89 of 21 7

APPENDIX 10 (C ontinued)

Group: G1 Dose: 0 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

24 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

15 F 1 1 1 1 1

16 F 1 1 1 1 1

27 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 F 1 1 1 1 1

29 1 M 1 C

- - -

2 F 1 2

- - -

30 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicable, C = Cannibalised, 1 = Normal, 2 = Dead

Note: Rat N° 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 90 of 21 7

APPENDIX 10 (C ontinued)

Group: G2 Dose: 10 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

46 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 85 85 85

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

47 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 1 1 1 1 1

9 M 1 1 1 1 1

10 M 2

- - - -

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

48 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 C

4 F 1 1 C

- -

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

49 1 M 1 C

- - -

2 M 1 C

- - -

3 M 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicable, C = Cannibalised, 1 = Normal, 2 = Dead, 85 = WeaknessJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 91 of 21 7

APPEN DIX 10 (C ontinued)

Group: G2 Dose: 10 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

50 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

15 F 1 1 1 1 1

51 1 M 1 1 1 1 1

2 F 1 1 1 1 1

3 F 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

52 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

15 F 1 2

- - -

16 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicable, 1 = Normal, 2 = De adJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 92 of 21 7

APPENDIX 10 (C ontinued)

Group : G2 Dose : 10 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

53 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

15 F 1 1 1 1 1

54 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 C

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

55 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 C

- - -

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

15 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicable, C = Cannibalised, 1 = Normal

Note: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 93 of 21 7

APPENDIX 10 (C ontinued)

Group: G2 Dose : 10 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

57 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

58 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

59 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

60 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

Key: M = Male, F = Female, 1 = NormalJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 94 of 21 7

APPENDIX 10 (C ontinued)

Group : G3 Dose : 100 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

78 1 M 1 1 1 1 1

2 M 1 C

- - -

3 F 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 C

- - -

79 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 85 85 85

8 M 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

81 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 C

- - -

82 1 F 1 1 1 1 1

2 F 1 1 1 1 1

3 F 1 1 1 1 1

4 F 2

- - - -

Key: M = Male, F = Female,

- = Not applicable, 1 = Normal

, C = Cannibalised, 2 = Dead, 85 = Weakness

Note: Rat N° 76, 77, 80 and 83 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 95 of 21 7

APPENDIX 10 (C ontinued)

Group : G3 Dose : 100 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

84 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 1 1 1 1 1

9 M 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

15 F 1 1 1 1 1

16 F 1 1 1 1 1

85 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

86 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

Key: M = Male, F = Female, 1 = Normal

Note: Rat N° 87 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 96 of 21 7

APPEND IX 10 (C ontinued)

Group: G3 Dose: 100 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

88 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 F 1 1 1 1 1

5 F 1 1 1 1 1

89 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

90 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 2

- -

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicable, 1 = Normal, 2 = DeadJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 97 of 21 7

APPENDIX 10 (C ontinued)

Group: G4 Dose: 1000 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

10 7 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

108 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 1 1 1 C

-

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

109 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 C

- - -

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

111 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 F 1 1 1 1 1

6 F 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicabl e, 1 = Normal

, C = Cannibalised

Note: Rat N° 106 and 110 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 98 of 21 7

APPENDIX 10 (C ontinued)

Group: G4 Dose: 1000 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

112 1 M 1 1 1 C

-

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 2

- - - -

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

14 F 1 1 1 1 1

15 F 1 1 1 1 1

115 1 M 1 1 C

- -

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

116 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

Key: M = Male, F = Female,

- = Not applicable, 1 = Normal, 2 = Dead

, C = Cannibalised

Note: Rat N° 113 and 114 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 99 of 21 7

APPENDIX 10 (C onti nued)

Group: G4 Dose: 1000 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

117 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 F 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

118 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 F 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

119 1 M 1 1 1 1 1

2 M 1 1 1 1 1

3 M 1 1 1 1 1

4 M 1 1 1 1 1

5 M 1 1 1 1 1

6 M 1 1 1 1 1

7 M 1 1 1 1 1

8 M 1 1 1 1 1

9 F 1 1 1 1 1

10 F 1 1 1 1 1

11 F 1 1 1 1 1

12 F 1 1 1 1 1

13 F 1 1 1 1 1

Key: M = Male, F = Female, 1 = Normal

Not e: Rat N° 120 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 100 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 11

Litter Size ( N°) and Sex Ratio (M : F ) of Individual Female

Group : G1 Dose : 0 mg/kg body weight/day

R at

N° Lactation Day 0 Lactation Day 4

N° of

Male

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F) N° of

Male

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F)

16 3 8 11 0.38 3 8 11 0.38

17 2 12 14 0.17 2 12 14 0.17

18 7 7 14 1.00 6 7 13 0 .86

19 13 1 14 13.00 12 1 13 12.00

20 2 1 3 2.00 2 1 3 2.00

21

- - - - - - - -

22 3 10 13 0.30 3 9 12 0.33

23 5 7 12 0.71 5 6 11 0.83

24 6 10 16 0.60 6 10 16 0.60

25

- - - - - - - -

26

- - - - - - - -

27 4 1 5 4.00 4 1 5 4.00

28

- - - - - - - -

29 1 1 2 1.00 0 0 0 -

30 4 6 10 0.67 4 6 10 0.67

Key:

- = Not applicable

Note: Rat N° 21, 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 101 of 217

APPENDIX 1 1 (Continued)

Group : G2 Dose : 10 mg/kg body weight/day

Rat

N° Lactation Day 0 Lactation Day 4

N° of

Male

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F) N° of

Male

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F)

46 4 6 10 0.67 4 6 10 0.67

47 10 3 13 3.33 9 3 12 3.00

48 3 8 11 0.38 2 7 9 0.29

49 3 4 7 0.75 1 4 5 0.25

50 5 10 15 0.50 5 10 15 0.50

51 1 4 5 0.25 1 4 5 0.25

52 7 9 16 0.78 7 8 15 0.88

53 6 9 15 0.67 6 9 15 0.67

54 7 7 14 1.00 6 7 13 0.86

55 6 9 15 0.67 5 9 14 0.56

56

- - - - - - - -

57 5 6 11 0.83 5 6 11 0.83

58 7 4 11 1.75 7 4 11 1.75

59 6 4 10 1.50 6 4 10 1.50

60 5 7 12 0.71 5 7 12 0.71

Key:

- = Not applicable

Note: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 102 of 217

APPENDIX 11 (Continued)

Group : G3 Dose : 1 00 mg/kg body weight/day

Rat

N° Lactation Day 0 Lactation Day 4

N° of

Male

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F) N° of

Ma le

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F)

76

- - - - - - - -

77

- - - - - - - -

78 2 8 10 0.25 1 7 8 0.14

79 8 6 14 1.33 8 6 14 1.33

80

- - - - - - - -

81 3 8 11 0.38 3 7 10 0.43

82 0 4 4 0.00 0 3 3 0.00

83

- - - - - - - -

84 9 7 16 1.29 9 7 16 1.29

85 5 4 9 1.25 5 4 9 1.25

86 5 9 14 0.56 5 9 14 0.56

87

- - - - - - - -

88 3 2 5 1.50 3 2 5 1.50

89 8 5 13 1.60 8 5 13 1.60

90 6 5 11 1.20 6 4 10 1.50

Key:

- = Not applicable

Note: Rat N° 76, 77, 80, 83 and 87 were non

- pregnant.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 103 of 217

APPENDIX 1 1 (Continued)

Group : G4 Dose : 1000 mg/kg body weight/day

Rat

N° Lactation Day 0 Lactation Day 4

N° of

Male

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F) N° of

Male

Pups N° of

Female

Pups Total

N° of

Pups Sex

Ratio

(M:F)

106

- - - - - - - -

107 5 6 11 0.83 5 6 11 0.83

108 8 4 12 2.00 7 4 11 1.75

109 5 8 13 0.63 5 7 12 0.71

110

- - - - - - - -

111 4 4 8 1.00 4 4 8 1.00

112 8 7 15 1.14 6 7 13 0.86

113

- - - - - - - -

114

- - - - - - - -

115 7 6 13 1.17 6 6 12 1.00

116 8 5 13 1.60 8 5 13 1.60

117 6 7 13 0.86 6 7 13 0.86

118 7 5 12 1.40 7 5 12 1.40

119 8 5 13 1.60 8 5 13 1.60

120

- - - - - - - -

Key:

- = Not applicable

Note: Rat N° 106, 110, 113, 114 and 120 were non

- pregnant.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 104 of 217



### Reproduction/Developmental Oral Toxicity Sc reening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 1 2

Body Weight (g) of Individual Pup s

Group : G1 Dose : 0 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

16 1 M 6.1 9.8

2 M 6 .0 10.5

3 M 6.2 9.5

4 F 6 .0 10.1

5 F 5.5 9.5

6 F 6 .0 9.7

7 F 6.1 10.3

8 F 6.1 9.3

9 F 5.8 10.1

10 F 5.6 10.1

11 F 5.8 9.7

17 1 M 6 .0 8.9

2 M 5.9 9.2

3 F 5.4 8.4

4 F 5.7 7.5

5 F 5.5 8.5

6 F 5.8 8 .0

7 F 5.6 8.6

8 F 5.4 8. 1

9 F 5.4 7.6

10 F 5.8 9.2

11 F 5.4 8.6

12 F 5.2 8.1

13 F 5.6 8.6

14 F 5 .0 8.1

18 1 M 5.3 9.7

2 M 5.8 9.6

3 M 5.8 10 .0

4 M 4.9 8.2

5 M 5.2 8.3

6 M 5.3 8.5

7 M

- -

8 F 5.2 8.6

9 F 4.7 7.2

10 F 4.9 8.9

11 F 4.8 8.9

12 F 4.9 8.6

13 F 4.7 8.3

14 F 4.5 7.4JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 105 of 217

APPENDIX 1 2 (C ontinued)

Group : G1 Dose : 0 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

19 1 M 5.5 9 .0

2 M 5.2 8.4

3 M 5.1 8.7

4 M 5.6 8.6

5 M 5.8 8.5

6 M 5.9 8.8

7 M 4.8 7.7

8 M 5 .0 8.8

9 M 5.1 8.4

10 M 4.9 7.8

11 M 5.1 8.5

12 M 5.5 8.9

13 M

- -

14 F 4.8 7.5

20 1 M 7.2 14.5

2 M 7.6 14.9

3 F 7 .0 14 .0

22 1 M 5.6 8.9

2 M 5.8 8.8

3 M 5.3 8.5

4 F 5.2 8.3

5 F 5.2 8.5

6 F 5.5 9 .0

7 F 5 .0

-

8 F 5.4 8.7

9 F 5.5 8.8

10 F 4.9 8 .0

11 F 4.9 7.5

12 F 5.4 8.7

13 F 5.7 8.6

23 1 M 6.1 11.6

2 M 6.9 12.7

3 M 6.4 11.5

4 M 6.6 11.3

5 M 5.2 5.2

6 F 6.2 11.2

7 F 5.5 11.1

8 F 5.8 11 .0

9 F 6.1 11.1

10 F 5.8 10.7

11 F 5.8 11.5

12 F

- -

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 21 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 106 of 217

APPENDIX 1 2 (C ontinued)

Group : G1 Dose : 0 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

24 1 M 5.7 8.7

2 M 5.3 8.2

3 M 4.5 7 .0

4 M 5 .0 7.5

5 M 5 .0 7.9

6 M 5.3 8.6

7 F 4.9 6.7

8 F 5 .0 7.4

9 F 5 .0 8.2

10 F 5.4 8.7

11 F 4.7 7 .0

12 F 5.1 7.8

13 F 5 .0 7.1

14 F 5 .0 7.9

15 F 4.8 6.9

16 F 4.5 6.8

27 1 M 7.5 14.4

2 M 7 .0 13.4

3 M 8 .0 14.4

4 M 7.5 14.9

5 F 7.3 14.2

29 1 M 6 .0

-

2 F 6 .0

-

30 1 M 6.5 10 .0

2 M 6.1 9.8

3 M 6.2 9.3

4 M 6.4 10.3

5 F 5.8 9.4

6 F 5.8 9.4

7 F 6.1 9.8

8 F 5.9 9.8

9 F 5.8 9.5

10 F 5.8 9.7

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 107 of 217

APPENDIX 1 2 (C ontinued)

Group : G 2 Dose : 10 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

46 1 M 7.3 12.7

2 M 7.1 12.4

3 M 7 .0 12.2

4 M 6.8 12.2

5 F 7.3 12.2

6 F 6.7 11.6

7 F 6.6 5.4

8 F 6.6 12.1

9 F 7 .0 11.7

10 F 6.5 11.8

47 1 M 4.8 7.3

2 M 4.7 6.6

3 M 5.3 8.2

4 M 4.7 7.2

5 M 4.9 8.3

6 M 4.8 7.4

7 M 5 .0 8.1

8 M 4.8 7.1

9 M 4.6 6.8

10 M

- -

11 F 5.1 8.2

12 F 5.3 8.2

13 F 4.9 7.4

48 1 M 6.2 10.7

2 M 6.6 10.7

3 M 6.4

-

4 F 5.8

-

5 F 6 .0 9.7

6 F 6.1 10.8

7 F 6.2 11 .0

8 F 5.4 9.4

9 F 6.5 10.4

10 F 5.8 10.1

11 F 5.7 9.7

49 1 M 5.9

-

2 M 5.9

-

3 M 6.1 11.8

4 F 6.8 10.3

5 F 5.8 10.7

6 F 5.5 13 .0

7 F 5.8 11.6

Key: M = Male, F = Female,

- = Not applicableJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 108 of 217

APPENDIX 1 2 (C ontinued)

Group : G 2 Dose : 10 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

50 1 M 5.9 9.6

2 M 5.8 9 .0

3 M 6.1 9.8

4 M 5.9 10

5 M 5.7 8.6

6 F 5.2 7.8

7 F 5.4 9.3

8 F 5.5 9.2

9 F 5.5 8.3

10 F 5.4 8 .0

11 F 6.1 9.6

12 F 5.3 9.2

13 F 5.3 8.8

14 F 5.7 8.5

15 F 5.3 8 .0

51 1 M 8.2 14 .0

2 F 7.2 12.4

3 F 7.4 13.3

4 F 6.9 9.6

5 F 7.2 13.2

52 1 M 5.7 9 .0

2 M 5.7 8.9

3 M 5.5 8.5

4 M 5.4 8 .0

5 M 5.3 8 .0

6 M 5.5 8.2

7 M 5.6 8.3

8 F 5.3 8 .0

9 F 5.4 8.6

10 F 4.8 7.1

11 F 5.6 8 .0

12 F 5.4 8.8

13 F 5.6 8.5

14 F 5 .0 7.5

15 F 5.2

-

16 F 5.1 7.4

Key: M = Male, F = Female,

- = Not applicableJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 109 of 217

APPENDIX 1 2 (C ontinued)

Group : G 2 Dose : 10 mg/kg body weight/da y

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

53 1 M 5.5 8.4

2 M 5.1 7.6

3 M 5.5 8.2

4 M 5.5 8.2

5 M 5.3 7.9

6 M 6 .0 7.7

7 F 5.1 7.7

8 F 4.9 7.7

9 F 5.1 8 .0

10 F 5 .0 7.6

11 F 5 .0 8.1

12 F 5.1 8.2

13 F 5.1 7.1

14 F 5.1 7.6

15 F 4.8 6.8

54 1 M 5.1 8 .0

2 M 5.3 6.8

3 M 6 .0

-

4 M 5.5 8.6

5 M 5.4 8.5

6 M 5.7 8.9

7 M 6 .0 9.8

8 F 6 .0 9.4

9 F 5.3 9 .0

10 F 5.2 8.3

11 F 5.9 9.2

12 F 5.3 9.3

13 F 5.2 8.5

14 F 6.4 10

55 1 M 5.1 8.4

2 M 4.9 7.6

3 M 5 .0 8. 6

4 M 3.8

-

5 M 5.7 8.5

6 M 5 .0 7.2

7 F 4.7 7.2

8 F 4.6 7.9

9 F 5.1 8 .0

10 F 4.5 7.7

11 F 4.2 6.7

12 F 4.6 6.9

13 F 5.5 8.4

14 F 4.9 8.1

15 F 5 .0 7.4

Key: M = Male, F = Female,

- = Not applicable

N ote: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 110 of 217

APPENDIX 1 2 (C ontinued)

Group : G 2 Dose : 10 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

57 1 M 5.8 9.3

2 M 5.7 9.4

3 M 5 .0 7.8

4 M 5.7 9.4

5 M 4.3 6.6

6 F 5.3 9 .0

7 F 5.1 8.3

8 F 5.4 8.7

9 F 5.7 9.2

10 F 4. 9 7.8

11 F 3.8 6.4

58 1 M 6 .0 9.4

2 M 6.3 9.8

3 M 6.3 10.1

4 M 6.3 10.2

5 M 6.7 10.3

6 M 6.7 11 .0

7 M 6.4 10.4

8 F 5.7 9.5

9 F 5.3 8.1

10 F 5.7 9.4

11 F 6.2 10 .0

59 1 M 5.8 9.6

2 M 6.4 10.6

3 M 6.6 10.7

4 M 6.1 10.2

5 M 6.4 10.5

6 M 5.9 10.1

7 F 6.1 9.9

8 F 5.4 9.2

9 F 5.9 9.9

10 F 5.9 9.8

60 1 M 5.7 9.7

2 M 6.5 9.1

3 M 6.1 9.3

4 M 6.3 9.2

5 M 5.1 7.6

6 F 6.4 8.9

7 F 6.4 8.3

8 F 5.9 9.6

9 F 6.1 9.4

10 F 5.9 8.9

11 F 5.6 8.9

12 F 6.2 9.4

Key: M = Male, F = Female,

- = Not applicableJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 111 of 217

APPENDIX 1 2 (C ontinued)

Group : G 3 Dose : 100 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

78 1 M 7.1 11.8

2 M 6.5

-

3 F 6.2 11.1

4 F 6.1 11.6

5 F 6.2 11.4

6 F 6 .0 11.1

7 F 5 .9 11.2

8 F 5.5 10.5

9 F 6.2 11.1

10 F 6.2

-

79 1 M 5.5 9.2

2 M 5.6 9.5

3 M 5.1 9 .0

4 M 5.2 8.5

5 M 5.5 9.1

6 M 5.2 8.4

7 M 5.7 5 .0

8 M 5.7 9.4

9 F 5.2 9.3

10 F 5 .0 8.4

11 F 4.7 7.6

12 F 5.1 8.8

13 F 5.4 8.6

14 F 5.5 9.5

81 1 M 5.9 11.2

2 M 6.2 11.5

3 M 6 .0 11.4

4 F 5.7 10.3

5 F 5.8 11.2

6 F 5.7 10.6

7 F 5 .0 9.2

8 F 5.8 10.7

9 F 5.5 9.8

10 F 5.2 9.9

11 F 4.9

-

82 1 F 6.9 12.1

2 F 7 .0 12.1

3 F 6.4 11.1

4 F

- -

Key: M = Male, F = Female,

- = Not a pplicable

Note: Rat N° 76, 77 and 80 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 112 of 217

APPENDIX 1 2 (C ontinued)

Group : G 3 Dose : 100 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

84 1 M 5.8 9.1

2 M 6.2 9.8

3 M 5.8 10 .0

4 M 5.7 9.2

5 M 5.7 9.4

6 M 6 .0 10.2

7 M 6.1 9.6

8 M 6.2 9.7

9 M 5.2 7.8

10 F 5.3 9.4

11 F 5.7 8.8

12 F 5.1 8.1

13 F 5.6 8.9

14 F 5.8 8.9

15 F 5.3 8.4

16 F 6.4 5.8

85 1 M 6.5 11.3

2 M 5.7 9.8

3 M 6 .0 10.4

4 M 6 .0 9.9

5 M 6.2 11 .0

6 F 5.7 8.2

7 F 5.3 9. 6

8 F 6.1 9.5

9 F 5.7 10 .0

86 1 M 6 .0 8.5

2 M 5.3 8.4

3 M 6.2 9.2

4 M 6.1 9.3

5 M 5.7 8.6

6 F 5.2 7.9

7 F 5.3 8.2

8 F 5.7 8 .0

9 F 5.5 8.7

10 F 5.2 8.4

11 F 5.5 8.5

12 F 5.6 8 .0

13 F 5.5 7.9

14 F 5.6 8.5

Key: M = Male, F = Fe male,

- = Not applicable

Note: Rat N° 83 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 113 of 217

APPENDIX 1 2 (C ontinued)

Group : G 3 Dose : 100 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

88 1 M 7.6 13.6

2 M 6.9 12.1

3 M 7.5 13 .0

4 F 7.5 10.2

5 F 7.1 12.5

89 1 M 6 .0 8.6

2 M 5.3 8.5

3 M 5.4 8.4

4 M 5.6 8.4

5 M 5.8 9.3

6 M 5.6 8.7

7 M 5.4 8.1

8 M 5.4 8.5

9 F 5.4 8.5

10 F 5 .0 8.6

11 F 4.8 7.6

12 F 5.5 8 .0

13 F 5.6 7.9

90 1 M 5.3 9 .0

2 M 5.8 9.6

3 M 5.3 9.2

4 M 5.6 9.5

5 M 5.3 8.9

6 M 5.2 8.8

7 F 5.1 9.1

8 F 5.4

-

9 F 5.2 9.1

10 F 5 .0 8.9

11 F 5.1 9.2

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 87 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 114 of 217

APPENDIX 1 2 (C ontinued)

Group : G 4 Dose : 1000 mg/kg body weight/day

R at N° Pup N° Sex Lactation Day 0 Lactation Day 4

107 1 M 6.7 10.7

2 M 6.6 10.8

3 M 6.9 10.3

4 M 6.5 10.7

5 M 6.7 10.5

6 F 6.8 11.2

7 F 6.3 10.2

8 F 6.8 10.9

9 F 6.4 10.2

10 F 6.3 10.1

11 F 6.2 8.3

108 1 M 5.8 9.5

2 M 5.9 9.3

3 M 6 .0 9.5

4 M 5.9 9.4

5 M 6.1 9.6

6 M 5.9 9.9

7 M 5.9 9.3

8 M 5.7

-

9 F 5.4 8.7

10 F 5.7 9.4

11 F 5.3 8.7

12 F 5.4 8.9

109 1 M 5.6 9.1

2 M 5.9 9.4

3 M 5.6 9.1

4 M 5.9 9.7

5 M 5.5 8.8

6 F 5.7 9.4

7 F 5.8 9.2

8 F 5.5 8.1

9 F 5 .4 8.9

10 F 5.6

-

11 F 5.4 8.6

12 F 5.6 9.4

13 F 5.5 9.3

111 1 M 5.5 11 .0

2 M 6 .0 11.1

3 M 6.1 11.3

4 M 5.4 10.3

5 F 5.4 10.8

6 F 5.7 10.9

7 F 5.4 10.1

8 F 6 .0 10.4

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 106 and 110 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 115 of 217

APPENDIX 1 2 (C ontinued)

Group : G 4 Dose : 1000 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

112 1 M 5.8

-

2 M 6.1 8.8

3 M 5.8 9.1

4 M 6 .0 8.5

5 M 6.1 9.7

6 M 5.6 8.5

7 M 5.5 8.7

8 M

- -

9 F 5.8 9.3

10 F 5.8 8.4

11 F 5.6 8.3

12 F 6 .0 8.8

13 F 5.9 8.9

14 F 6.1 9.6

15 F 4.3 5.5

115 1 M 6 .0

-

2 M 6.6 10.9

3 M 6.6 11.4

4 M 6.3 10

5 M 6.2 10.6

6 M 6.7 11.6

7 M 6.6 11.3

8 F 6.3 10.8

9 F 6.4 11.2

10 F 6.4 11.3

11 F 6.1 10.9

12 F 6.3 10.4

13 F 6 .0 10.2

116 1 M 5.8 11 .0

2 M 6.3 10.6

3 M 6.3 10.5

4 M 6.7 10.2

5 M 5.4 7 .0

6 M 6.2 10.7

7 M 6 .0 11.2

8 M 6.1 11 .0

9 F 6 .0 10.7

10 F 5.8 10.7

11 F 6.1 11.1

12 F 6.3 11.4

13 F 6 .0 11 .0

Key: M = M ale, F = Female,

- = Not applicable

Note: Rat N° 113 and 114 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 116 of 217

APPENDIX 1 2 (C ontinued)

Group : G 4 Dose : 1000 mg/kg body weight/day

Rat N° Pup N° Sex Lactation Day 0 Lactation Day 4

117 1 M 7.4 10.5

2 M 6.4 10.9

3 M 6.2 10.7

4 M 5 .6 11.2

5 M 6.5 9.5

6 M 5.9 10.3

7 F 6 .0 8.9

8 F 6.5 10.5

9 F 5.8 9.7

10 F 5.3 10 .0

11 F 5.7 10 .0

12 F 5.5 10.6

13 F 6 .0 9.6

118 1 M 6 .0 9.8

2 M 6.3 10.6

3 M 6.2 10.3

4 M 5.2 9.8

5 M 6.7 10.3

6 M 6.3 10.6

7 M 6.3 10.7

8 F 5 .7 9.2

9 F 6 .0 10.2

10 F 5.4 9.4

11 F 5.7 10

12 F 5.8 9.5

119 1 M 5.6 9.5

2 M 5.9 9.9

3 M 5.6 9 .0

4 M 5.3 8.6

5 M 5.7 9.4

6 M 5.9 10.2

7 M 5.9 9.5

8 M 5.9 10 .0

9 F 5.2 8.2

10 F 5.3 8.8

11 F 4.8 8.3

12 F 5.7 9.3

13 F 5.4 9.1

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 120 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 117 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 1 3

Mean Pup s Body wei ght (g) of Individual Female

Group : G1 Dose : 0 mg/kg body weight/day

Rat N° Sex Lactation Day

Sex Lactation Day

Sex Lactation Day

0 4 0 4 0 4

16 M 6.10 9.93 F 5.86 9.85 MF 5.93 9.87

17 M 5.95 9.05 F 5.48 8.28 MF 5.55 8.39

18 M 5.38 9.05 F 4.81 8 .27 MF 5.08 8.63

19 M 5.29 8.51 F 4.80 7.50 MF 5.25 8.43

20 M 7.40 14.70 F 7.00 14.00 MF 7.27 14.47

21

- - - - - - - - -

22 M 5.57 8.73 F 5.27 8.46 MF 5.34 8.53

23 M 6.24 10.46 F 5.87 11.10 MF 6.04 10.81

24 M 5.13 7.98 F 4.94 7.45 MF 5.01 7.65

25

- - - - - - - - -

26

- - - - - - - - -

27 M 7.50 14.28 F 7.30 14.20 MF 7.46 14.26

28

- - - - - - - - -

29 M 6.00 - F 6.00

- MF 6.00

-

30 M 6.30 9.85 F 5.87 9.60 MF 6.04 9.70

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 21, 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 118 of 217

APPENDIX 1 3 (Continued)

Group : G2 Dose : 10 mg/kg body weight/day

Rat N° Sex Lactation Day

Sex Lactation Day

Sex Lactation Day

0 4 0 4 0 4

46 M 7.05 12.38 F 6.78 10.80 MF 6.89 11.43

47 M 4.84 7.44 F 5.10 7.93 MF 4.91 7.57

48 M 6.40 10.70 F 5.94 10.16 MF 6.06 10.28

49 M 5.97 11.80 F 5.98 11.40 MF 5.97 11.48

50 M 5.88 9.40 F 5.47 8.67 MF 5.61 8.91

51 M 8.20 14.00 F 7.18 12.13 MF 7.38 12.50

52 M 5.53 8.41 F 5.27 7.99 MF 5.38 8.19

53 M 5.48 8.00 F 5.02 7.64 MF 5.21 7.79

54 M 5.57 8.43 F 5.61 9.10 MF 5.59 8.79

55 M 4.92 8.06 F 4.79 7.59 MF 4.84 7.76

56

- - - - - - - - -

57 M 5.30 8.50 F 5.03 8.23 MF 5.15 8.35

58 M 6.39 10.17 F 5.73 9.25 MF 6.15 9.84

59 M 6.20 10.28 F 5.83 9.70 MF 6.05 10.05

60 M 5.94 8.98 F 6.07 9.06 MF 6 .02 9.03

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 119 of 217

APPENDIX 1 3 ( Continued)

Group : G3 Dose : 1 00 mg/kg body weight/day

Rat N° Sex Lactation Day

Sex Lactation Day

Sex Lactation Day

0 4 0 4 0 4

76

- - - - - - - - -

77

- - - - - - - - -

78 M 6.80 11.80 F 6.04 11.14 MF 6.19 11.23

79 M 5.44 8.51 F 5.15 8.70 MF 5.31 8.59

80

- - - - - - - - -

81 M 6.03 11.37 F 5.45 10.24 MF 5.61 10.58

82 M - - F 6.77 11.77 MF 6.77 11.77

83

- - - - - - - - -

84 M 5.86 9.42 F 5 .60 8.33 MF 5.74 8.94

85 M 6.08 10.48 F 5.70 9.33 MF 5.91 9.97

86 M 5.86 8.80 F 5.46 8.23 MF 5.60 8.44

87

- - - - - - - - -

88 M 7.33 12.90 F 7.30 11.35 MF 7.32 12.28

89 M 5.56 8.56 F 5.26 8.12 MF 5.45 8.39

90 M 5.42 9.17 F 5.16 9.08 MF 5.30 9.13

Ke y: M = Male, F = Female,

- = Not applicable

Note: Rat N° 76, 77, 80 , 83 and 87 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 120 of 217

APPENDIX 1 3 (Continued)

Group : G4 Dose : 1000 mg/kg body weight/day

Rat N° Sex Lactation Day

Sex Lactation Day

Sex Lactation Day

0 4 0 4 0 4

106

- - - - - - - - -

107 M 6.68 10.60 F 6.47 10.15 MF 6.56 10.35

108 M 5.90 9.50 F 5.45 8.93 MF 5.75 9.29

109 M 5.70 9.22 F 5.56 8.99 MF 5.62 9.08

110

- - - - - - - - -

111 M 5.75 10.93 F 5.63 10.55 MF 5.69 10.74

112 M 5.84 8.88 F 5.64 8.40 MF 5.74 8.62

113

- - - - - - - - -

114

- - - - - - - - -

115 M 6.43 10.97 F 6.25 10.80 MF 6.35 10.88

116 M 6.10 10.28 F 6.04 10.98 MF 6.08 10.55

117 M 6.33 10.52 F 5.83 9.90 MF 6.06 10.18

118 M 6.14 10.30 F 5.72 9.66 MF 5.97 10.03

119 M 5.73 9.51 F 5.28 8.74 MF 5 .55 9.22

120

- - - - - - - - -

Key: M = Male, F = Female,

- = Not applicable

Note: Rat N° 1 06, 110, 113, 114 and 120 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 121 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68- 4; US EPA HPV 3) in Wistar Rats

APPENDIX 1 4

Pre and Postnatal Data

Group: G1 Dose: 0 mg/kg body weight/day

Rat

N° Number of Pre

- implantation Post

- implantation Post

- natal

Corpora

lutea Implant

ation Live

Pups on

Lac.

Day 0 Live

Pups on

Lac

. Day 4 Loss Loss

(% ) Loss Loss

(%) Loss Loss

(%)

16 13 13 11 11 0 0.0 2 15.4 0 0.0

17 15 14 14 14 1 6.7 0 0.0 0 0.0

18 15 15 13 13 0 0.0 2 13.3 0 0.0

19 14 14 13 13 0 0.0 1 7.1 0 0.0

20 10 4 3 3 6 60.0 1 25.0 0 0.0

21

- - - - - - - - - -

22 13 13 13 12 0 0.0 0 0.0 1 7 .7

23 15 13 11 11 2 13.3 2 15.4 0 0.0

24 16 16 16 16 0 0.0 0 0.0 0 0.0

25

- - - - - - - - - -

26

- - - - - - - - - -

27 12 5 5 5 7 58.3 0 0.0 0 0.0

28

- - - - - - - - - -

29 9 2 2 0 7 77.8 0 0.0 2 100.0

30 12 12 10 10 0 0.0 2 16.7 0 0.0

Key: L ac. = Lactation

, - = Not applicable

Note: Rat N° 21, 25, 26 and 28 were non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 122 of 217

APPENDIX 14 (Continued)

Group: G2 Dose: 10 mg/kg body weight/day

Rat

N° Number of Pre

- implantation Post

- implantation Post

- natal

Corpora

lutea Implant

ation Live

Pups o n

Lac.

Day 0 Live

Pups on

Lac.

Day 4 Loss Loss

(%) Loss Loss

(%) Loss Loss

(%)

46 12 11 10 10 1 8.3 1 9.1 0 0.0

47 15 13 12 12 2 13.3 1 7.7 0 0.0

48 12 12 11 9 0 0.0 1 8.3 2 18.2

49 9 8 7 5 1 11.1 1 12.5 2 28.6

50 16 15 15 15 1 6.3 0 0.0 0 0.0

51 12 6 5 5 6 50.0 1 16.7 0 0.0

52 16 16 16 15 0 0.0 0 0.0 1 6.3

53 15 15 15 15 0 0.0 0 0.0 0 0.0

54 14 14 14 13 0 0.0 0 0.0 1 7.1

55 16 15 15 14 1 6.3 0 0.0 1 6.7

56

- - - - - - - - - -

57 13 13 11 11 0 0.0 2 15.4 0 0.0

58 12 11 11 11 1 8.3 0 0.0 0 0.0

59 12 11 10 10 1 8.3 1 9.1 0 0.0

60 15 13 12 12 2 13.3 1 7.7 0 0.0

Key: Lac. = Lactation

, - = Not applicable

Note: Rat N° 56 was non

- pregnantJRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 123 of 217

APPENDIX 14 (Continued)

Group: G3 Dose: 1 00 mg/kg body weight/day

Rat

N° Number of Pre

- implantation Post

- implantation Post

- natal

Corpora

lutea Implant

ation Live

Pups on

Lac.

Day 0 Live

Pups on

Lac.

Day 4 Loss Loss

(%) Loss Loss

(%) Loss Loss

(%)

76

- - - - - - - - - -

77

- - - - - - - - - -

78 17 13 10 8 4 23.5 3 23.1 2 20.0

79 14 14 14 14 0 0.0 0 0.0 0 0. 0

80

- - - - - - - - - -

81 15 11 11 10 4 26.7 0 0.0 1 9.1

82 11 5 3 3 6 54.5 2 40.0 0 0.0

83

- - - - - - - - - -

84 17 16 16 16 1 5.9 0 0.0 0 0.0

85 11 11 9 9 0 0.0 2 18.2 0 0.0

86 15 14 14 14 1 6.7 0 0.0 0 0.0

87

- - - - - - - - - -

88 6 5 5 5 1 16.7 0 0.0 0 0.0

89 15 14 13 13 1 6.7 1 7.1 0 0.0

90 12 12 11 10 0 0.0 1 8.3 1 9.1

Key: Lac. = Lactation

, - = Not applicable

Note: Rat N° 76, 77, 80, 83 and 87 were non

- pregnant

.JRF Study Number: 496 -1- 04

- 4306 ( Final Report) Page 124 of 217

APPENDIX 14 (Continued)

Group: G4 Dose: 1000 mg/kg body weight/d ay

Rat

N° Number of Pre

- implantation Post

- implantation Post

- natal

Corpora

lutea Implant

ation Live

Pups on

Lac.

Day 0 Live

Pups on

Lac.

Day 4 Loss Loss

(%) Loss Loss

(%) Loss Loss

(%)

106

- - - - - - - - - -

107 13 13 11 11 0 0.0 2 15.4 0 0.0

108 12 12 12 11 0 0.0 0 0.0 1 8.3

109 13 13 13 12 0 0.0 0 0.0 1 7.7

110

- - - - - - - - - -

111 9 8 8 8 1 11.1 0 0.0 0 0.0

112 15 15 14 13 0 0.0 1 6.7 1 7.1

113

- - - - - - - - - -

114

- - - - - - - - - -

115 16 16 13 12 0 0.0 3 18.8 1 7.7

116 14 14 13 13 0 0.0 1 7.1 0 0.0

117 15 13 13 13 2 13.3 0 0.0 0 0.0

118 17 13 12 12 4 23.5 1 7.7 0 0.0

119 13 13 13 13 0 0.0 0 0.0 0 0.0

120

- - - - - - - - - -

Key: Lac. = Lactation

, - = Not applicable

Note: Rat N° 106, 110, 113, 114 and 120 were non

- pregnant.JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 125 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3

- nitrobenzene Sulfonate (CAS # 127 -68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 15

Dose Formulation Analysis ReportJRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 126 of 217

APPENDIX 15 (continued )

10 30 50 70 90 110

Concentration (mg/L) 0.4 1.4 2.4 3.4 4.4Mean Peak Area (Millions)

Intercept with y-axis (a) = -17111.65

Slope of the line (b) = 42129.93

Correlation co-efficient or 'r' value = 0.999

Equation : Y = bX + a

Y = 42129.93X + (-17111.65) 

### 3. EXPERIMENTAL PROCEDURE

Analysis on 10/12/2012

3.1 Preparation of Sodi um 3

- Nitrobenzensulfonate Reference Standard Stock Solution

A quantity of 10 3. 84 mg Sodium 3

- Nitrobenzensulfonate reference standard (9 8.0 % purity) was

weighed in to a volumetric flask of 1 0 0 mL capacity, dissolved in 5 0 .0 mL RO water and the volume

was made upto the mark with RO water. The concentration of Sodium 3

- Nitrobenzensulfonate in the

stock solution was 1 017.63 mg/L ( RS )

.

3.2 Preparation of Sodium 3

- Nitrobenzensulfonate Reference Standard Working Solution s for

Linear Dynamic Range

I dentification

of Reference

Standard

Solution Solution

Taken

(mL) Final

Volume

(mL) Volume Made

With Obtained

Concentration

( mg /L) Identification

of Reference

Standard

Solution

( 1017.63 mg/ L)

RS 0.1 10

RO water 10.18 L1

0.2 10 20.35 L2

0.4 10 40.71 L3

0.6 10 61. 06 L4

0.8 10 81.41 L5

1.0 10 101.76 L 6

The Reference Working Solutions ( L1, L2, L3, L4, L5 and L6 ) were injected onto the HPLC in

duplicate and the mean peak area was plotted against concentration ( m g/L ). The correlation coefficient

(r), slope (b) and intercept (a) were calculated.

3. 3 Linear Dynamic Curve

Figure 1

Page 2 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 127 of 217

APPENDIX 15 (continued )

10 30 50 70 90 110

Concentration (mg/L) 0 1 2 3 4 5Mean Peak Area (Millions)

Intercept with y-axis (a) = -3921.96

Slope of the line (b) = 39432.97

Correlation co-efficient or 'r' value = 0.999

Equation : Y = bX + a

Y = 39432.97X + (-3921.96) 

### Analysis on 04/01/2013

3.4 Preparation of Sodium 3

- Nitrobenzensulfonate Reference Standard Stock Solution

A quantity of 102.50 mg Sodium 3

- Nitroben zensulfonate reference standard (9 8.0 % purity) was

weighed into a volumetric flask of 1 0 0 mL capacity, dissolved in 5 0 .0 mL RO water and the volume

was made upto the mark with RO water. The concentration of Sodium 3

- Nitrobenzensulfonate in the

stock solut ion was 1004.50 mg/L ( RS1 )

.

3. 5 Preparation of Sodium 3

- Nitrobenzensulfonate Reference Standard Working Solutions for

Linear Dynamic Range

I dentification

of Reference

Standard

Solution Solution

Taken

(mL) Final

Volume

(mL) Volume Made

With Obtained

Concen tration

( mg /L) Identification

of Reference

Standard

Solution

( 1004.50 mg/ L)

RS1 0.1 10

RO water 10.05 L1

0.2 10 20.09 L2

0.4 10 40.18 L3

0.6 10 60.27 L4

0.8 10 80.36 L5

1.0 10 100.45 L6

The Reference Working Solutions ( L1, L2, L3, L4, L5 and L6 ) were injected onto the HPLC in

duplicate and the mean peak area was plotted against concentration ( mg/L ). The correlation coefficient

(r), slope (b) and intercept (a) were calculated.

3. 6 Linear Dynamic Curve

Figure 2

Page 3 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 128 of 217

APPENDIX 15 (continued )

3. 7 Sample Preparation on 10/12/2012 and 04/01/2013

A volume of 0.5 mL from 1 mg/mL solution ( G2R1, G2R2, G2R3, G2R4, G2R5 and G2R6) were

transferred into separate volumetric flasks of 10 mL capacity and the volume of each flask was made

up to the mark with RO water. The dilution factor was 20.

A volume of 1.0 mL from 10 mg/mL solution ( G3R1, G3R2, G3R3, G3R4, G3R5 and G3R6) were

transferred into separate volumetric flasks of 10 mL capacity and the volume of each flask was made

up to the mark with RO water. A volume of 0.5 mL from these solution were transferred into separate

volumetric flasks of 10 mL capacity and the volume of each flask was made up to the mark with RO

water The dilution factor was 200.

A volume of 0.1 mL from 100 mg/mL solution ( G4R1, G4R2, G4R3, G4R4, G4R5 and G4R6) were

transferred into separate volumetric flasks of 10 mL capacity and the volume of each flask was made

up to the mark with RO water. A volume of 0.5 mL from these solution were transferred into separate

volumetric f lasks of 10 mL capacity and the volume of each flask was made up to the mark with RO

water The dilution factor was 2000.

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

.

4. Calculation

The active ingredient concen tration (X) was calculated using the following formula:

Y = b X + a

Y - a A.I. concentration ( m g/L ) X =

------- x D

b

Y = Peak area of the sample

a = Intercept

b = Slope

D = Dilution factor

The % Recovery was calculated using the following formula:

Detected concentration

% Recovery =

----------------------------------------- x 100

Theoretical concentration in

RO water based on p urity of test item

Page 4 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 129 of 217

APPENDIX 15 (continued )

4. RESULTS

Result of Active Ingredient Concentration and Homogeneity of Sodium 3

- Nitrobenzensulfonate

in RO water

(10/12/2012)

Group and

Concentration

(mg/mL ) Theoretical

Concentration

in Test media

based on P urity

of Test Item

(mg/L) Sodium 3

- Nitrobenzensulfonate

Detected

Concentration

(mg/L) Mean

Concent

ration

(mg/L) %

Recovery Mean

Recovery

(%) SD %RSD

G1

(Control) 0.0 ND

- -

- - ND

-

ND

-

ND

-

ND

-

ND

-

G2

(1.0) 969 931.34

932.33 96.11

96.21 3.9748 0.43 929.96 95.97

929.85 95.96

934.32 96.42

939.49 96.95

929.01 95.87

G3

(10.0) 9690 9307.01

9341.05 96.05

96.40 55.6336 0.60 9288.02 95.85

9352.19 96.51

9409.08 97.10

9402.98 97.04

9287.04 95.84

G4

(100.0) 96900 93419.22

93517.90 96.41

96.51 246.3229 0.26 93150.43 96.13

93423.12 96.41

93690.43 96.69

93861.14 96.86

93563.06 96.56

Key: ND = Non Detectable (< LO Q = 0. 5 m g/L)

Purity of Test Item = 9 6.9 %

Page 5 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 130 of 217

APPENDIX 15 (continued )

(04/01/2013)

Group and

Concentration

(mg/mL) Theoretical

Concentration

in Test media

based on Purity

of Test Item

(mg/L) Sodium 3

- Nitrobenzensulfonate

Detected

Concentration

(mg/L) Mean

Concent

ration

(mg/L) %

Recovery Mean

Recovery

(%) SD %RSD

G1

(Control) 0.0 ND

- -

- - - ND

-

ND

-

ND

-

ND

-

ND

-

G2

(1.0) 969 956.58

954.40 98.72

98.49 1.8084 0.19 953.62 98.41

954.02 98.45

954.41 98.49

951.61 98.21

956.15 98.67

G3

(10.0) 9690 9481.28

9500.12 97.85

98.04 14.9474 0.16 9519.02 98.24

9485.74 97.89

9497.16 98.01

9513.23 98.18

9504.31 98.08

G4

(100.0) 96900 94019.44

94025.65 97.03

97.04 246.2356 0.26 94308.69 97.33

94343.89 97.36

93857.90 96.86

93845.73 96.85

93778.22 96.78

Key: ND = Non Detectable (< LO Q = 0.5 mg/L)

Purity of Test Item = 96.9 %

Page 6 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 131 of 217

APPENDIX 15 (continued )

6. Typical Chromatograms

Page 7 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 132 of 217

APPENDIX 15 (continued )

Page 8 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 133 of 217

APPENDIX 15 (continued )

Page 9 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 134 of 217

APPENDIX 15 (continued )

Page 10 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 135 of 217

APPENDIX 15 (continued )

Page 11 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 136 of 217

APPENDIX 15 (continued )

Page 1 2 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 137 of 217

APPENDIX 15 (continued )

Page 13 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 138 of 217

APPENDIX 15 (continued )

Page 14 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 139 of 217

APPENDIX 15 (continued )

Pa ge 15 of 16JRF Study Number: 496 -1- 04

- 4306 (Final Report) Page 140 of 217

APPENDIX 15 (continued )

Page 16 of 16

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 142 of 217

APPENDIX 16 (Continued)

PERSONNEL INVOLVED

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

Stu dy Personnel : DR. CHINTAMANI CHAVAN, M.V.Sc.

DR. MAHESH BRAHMANKAR, M.V.Sc.

BHAVNA PRAJAPATI, B.Sc ., D.M.L.T.

PRAVINA PATEL, B.Sc., D.M.L.T.

DHIRUBHAI NAIK, B.A

.

KOMAL MANIAR, M. Sc

.

NITIGNA PATEL, B.Sc. , D.M.L.T.

HELI RANA, M.Sc.

JOHN MOSES, M.Sc. , P.G.D.C. S. A.

PAR T HA BOSE, B.Com. , P.G.D.C.A.

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 143 of 217

APPENDIX 16 (Continued)

CONTENTS

Page N°

TITLE PAGE ........................................................................................................................................... 141

PERSONNEL INVOLVED ...................................................................................................................... 142

CONTENTS .............................................................................................................................................. 143

LIST OF ABBREVIATIONS ................................................................................................................... 144

1. EXPERIMENTAL PROCEDURE .............................................................................................. 145

2. RESULTS AND DISCUSSION .................................................................................................. 145

2.1 Terminal Body Weight (g), Absolute Organ Weight (g) and Organ Weight Relative to

Terminal Body Weight (%) Parental Rat ........................................................................ 145

2.2 Macroscopic Findings Parental Rat ............................................................................... 145

2.3 Macroscopic Findings F1 Preweanling (4 th 

### Day) Pups ................................................... 146

2.4 Microscopic Findings Parental Rat ................................................................................ 146

3. C ONCLUSION ............................................................................................................................ 147

4. REFERENCES ............................................................................................................................. 147

TABLES

A. Terminal Body Weight , Absolute Organ Weight (g) and Organ Weight Relative to

Terminal Body Weight (%) - Group Mean Values Parental Rat ................................... 148

B. Gross and Microscopic Findings – Summary by Groups Parent al Rat ........................... 149

C. Gross Findings – Summary by Groups F1 Preweanling (4 th 

### Day) Pups ......................... 150

APPENDICES

A. Terminal Body Weight (g) , Absolute Organ Weight (g) and Organ Weight Relative to

Terminal Body Weig ht (%) Parental Rat ........................................................................ 151

B. Macroscopic and Microscopic Findings of Individual Rat - Parental Rat ...................... 155

C. Gross Findings of Individual F1 Preweanling (4 th 

### Day) Pups ....................................... 163

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 144 of 217

APPENDIX 16 (Continued)

LIST OF ABBREVIATIONS

- - Significantly higher than control (p 0.05)

 ̄ - Significantly lower than control (p 0.05)

- - - Significantly higher than control (p 0.01)

 ̄  ̄ - Significantly lower than control (p 0.01)

% - Percentage

b.wt. - Body Weight

F - Female

FD - Found dead

g - Gram

G1 - Control (0 mg/kg b.wt./day)

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

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

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

kg - Kilogram

M - Male

MNC - - Mononuclear C ell

mg - Milligram

mL

- Milliliter

N - Number of Anima ls

N° - Number

NAD

- No Abnormality Detected

SD - Standard Deviation

X

- Organs not examined as per study plan, except where gross lesions were recorded

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 145 of 217

APPENDIX 16 (Continued)

1. EXPERIMENTAL PROCEDURE

At scheduled sacr ifice dates, all the rats belonging to various experimental groups (G1 to G4) were

euthanised by carbon dioxide asphyxiation and pups were euthanised by intraperitoneal injection of

thiopen tone sodium and subjected to gross necropsy under the direct superv ision of a veterinary

pathologist . First the carcasses (all rats and pups) were examined for external abnormalities

followed by thorough examination of organs by cut opening the thoracic , abdominal and cranial

cavities

. Special attention was paid to organ of the reproductive systems.

V agina, uterus with oviducts, cervix, seminal vesicles with coagulating gland and prostate from all

the rats (G1 to G4) were preserved in 10% neutral buffered formalin. Ovaries, testes and

epididymis were preserved in Bouin’s fixative. Testes and epididymis from male rats were weighed

and relative weights (organ weight relative to terminal body weight) were calculated

.

Ovaries and epididymis were processed routinely to have H & E stained sections

. Testes were

processed to have H & E and PAS stained sections from the control (G1) and high dose (H4)

groups . The prepared slides were examined under microscope by the Study Pathologist to perform

detail histopatholog ical examination of the ovaries, testes and epididymis from the cont ro l and high

dose groups.

Last date of slide reading: April 19, 2013.

2. RESULTS AND DISCUSSION

2.1 Terminal Body Weight (g) , Absolute Organ Weight (g) and Organ Weight R elative to

Terminal Body Weight (%) - Parental Rat [Table A and Appendix A ]

The t erminal body weight and organ weight (both absolute and relative weight of testes and

epididymis ) in male rats belonging to treatment groups (G2 to G4) were comparable to the control

(G1) group except statistically significant lower absolute weight of epid idymes in mid dose group

(G3) when compared to the control group (G1)

. This variation in epididymes weight in mid dose

group (G3) was considered incidental and non

- specific in nature and not related to the treatment

with the test item due to lack of dose dependency.

2.2 Macroscopic Findings

- Parental Rat s [Table B and Appendix B ]

External

External examination of the terminally sacrificed rats of all the experimental groups (G1 to G4 ) did

not reveal any lesion of pathological significance.

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 146 of 217

APPENDIX 16 (Continued)

Interna l

Internal examination of the terminally sacrificed anima ls of all the groups did not reveal any lesio n

of pathological significance except reduction of size of right testes (G3M: 01/15) and white nodule

in cauda of right epididymes in one animal (G4M: 01/15)

.

2.3 Macroscopic Findings

- F1 Preweanling (4 th 

### Day) Pups [Table C and Appendix C ]

External

External examination of the terminally sacrificed as well as deceased pups belonging to different

experimental groups (G1 to G4) did not reveal any lesi o n of pathological significance except

emaciation in one female pup (G2: 01/87) and one male pup ( G3: 01/48 ).

Internal

Internal examination of the terminally sacrificed pups of different experimental groups (G1 to G4)

did not reveal any lesion of pathologic al significance . However, the deceased pups revealed various

lesions viz. congestion in lungs (G1M: 01/ 49 , G1F: 02/6 4, G2M: 01/69, G2F: 0 1 /8 7 , G3F: 0 2/56

,

G4M: 1/6 3), congestion in liver ( G1M: 01/ 49 , G1F: 01/ 64, G2F: 01 /87 , G3F: 0 1/56) and pallor in

kidney s ( G2F: 01/88 ). These are spontaneous/incidental in nature and considered to be of no

toxicological relevance.

2.4 Microscopic Findings

– Parental Rat [Table B and Appendix B ]

Detailed histological examinations performed on the ovaries, testes and epidi dymides of control and

high dose groups (G1 and G4). Testicular examination was carried out with special emphasis on

sperm staging and interstitial testicular cell morphology. Histological examination of epididymis

included the caput, corpus and cauda port ion. Ovaries were examined for qualitative depletion of

the primordial follicle population. Detailed histological examinations of ovaries, testes and

epididymis from the control and high dose groups (G1 and G4) did not show any lesion of

pathological signi ficance.

Microscopic examination revealed varying degree of pathological changes in different organs of

control and high dose groups (G1 and G4). The lesions observed included: Testes ( interstitial

oedema, germ cells degeneration/exfoliati on) and Epididym ides (sperm granuloma, reduced

sperm s in tubular lumen and interstitial MNC infiltration ).

Multiple sperm granuloma were observed in the epididymes of one male rat (Animal No. 103 )

belonging to the high dose group (G4) resulting in reduced number of sperms in the lumen of the

epididymal ducts. It was considered to be an isolated occurrence/incidental in nature and not

treatment related. Other microscopic lesions as mentioned above were observed at a lower rate of

occurrence in both control g roup (G1) and high dose group (G4) and were mostly minimal to mild

in nature, non specific and insignificant lacking consistency and specific linearity pattern.

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 147 of 217

APPENDIX 16 (Continued)

Hence, these lesions were considered to be spontaneous or incide ntal in nature without an

indi cation of an effect of treatment with the test item.

3. CONCLUSION

Thus it is concluded that the test item [Sodium 3 -n itrobenzene Sulfonate (CAS # 127 -68- 4; US

EPA HPV 3)] did not produce any adverse pathological alterations in testes, epididymis and ovaries

up to the dose level of 1000 mg/kg/ b. wt. / day given by oral route in the Wistar rats.

4. REFERENCES

Boorman, G. A., Eustis, S. L., Elwell, M. R., Montgomery, Jr., C. A. and MacKenzie, W. F. (1990).

Pathology of the Fischer Rat: Reference and Atlas. Eds., Academic Press, Inc. Harcourt Brace

Jovanovich Publishers, San Diego, New York, Boston, London, Sydney, Tokyo, Torento.

Greaves, P. and Faccini, J. M. (1992). Rat Histopathology, 2 nd 

### Edn, Elsevier, Amsterdam

–

London- New York

- Tokyo.

Russell, L. D., Ettlin, R. A., SinhaHikim, A. P. and Clegg, E.D. (1990). Histological and

Histopathological Evaluation of the Testes. 1 st 

### Edn. Cache River Press, FL, USA.

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 148 of 217

APPENDIX 16 (Continued)

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

Terminal Body Weight (%)

– Group Mean Values

– Parental Rat

Parameters Absolute organ weight (g) Organ weight r elative to terminal

body weight (%)

Group s G1 G2 G3 G4 G1 G2 G3 G4

Dose

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

N o 

### 15 15 15 15 15 15 15 15

Terminal

Body

Weight Mean 447.993 425.920 422.827 429.467 Not Applicable

SD 44.802 25.306 36.699 26.986 Not Applicable

Testes Mean 4.035 3.950 3.844 4.003 0.907 0.928 0.911 0.933

SD 0.353 0.321 0.464 0.320 0.105 0.065 0.093 0.066

Epididymis Mean 1.554 1.438 1.370

 ̄  ̄ 1.448 0.350 0.337 0.325 0.338

SD 0.113 0.130 0.174 0.109 0.040 0.023 0.040 0.029

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 149 of 217

APPENDIX 16 (Continued)

TABLE B : Gross and Microscopic Findings – Summary by Groups ( Parent al rats )

Keys: M = Male, F = Female

Note: i. Organs not included in table did not reveal any gross and histopathologic al findings.

ii. ‘X’ = Organs not examined as per study plan / sex difference, except where gross lesions

were recorded

.

Group s & Dose G1

0 mg/kg

b. wt./day G2

10 mg/kg

b. wt./da y G3

100 mg/kg

b. wt./day G4

1000 mg/kg

b. wt./day

Organs & Lesions Sex M F M F M F M F

N° of

Animals 15 15 15 15 15 15 15 15

GROSS FINDINGS

EXTERNAL EXAMINATION

No Abnormality Detected 15 15 15 15 15 15 15 15

INTERNAL EXAMINATION

Testes

Reduce d size 0 X 0 X 1 X 0 X

Epididymes

White nodule, cauda 0 X 0 X 0 X 1 X

MICROSCOPIC FINDINGS

Testes

Oedema, interstitial 1 X X X X X 1 X

Degeneration/ exfoliation, germ cells,

tubular 0 X X X 1 X 0 X

Epididymis

MNC infiltration, interstitial 1 X X X X X 1 X

Granuloma, sperm 0 X X X X X 1 X

Reduced sperms, tubular lumen 0 X X X X X 1 X

Ovaries

No Abnormality Detected X 15 X 15 X 15 X 15

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 150 of 217

APPENDIX 16 (Continued)

TABLE C: Gross Findings

– Summary by Groups F1 Preweanling ( 0 th 

### -4 th 

### Day) Pups

Keys: M = Male, F = Female

Note: i: Organs not included in table did not reveal any gross findings.

ii: Non Pregnant female rats in G1, G2, G3 and G4 were not included in Dam. Group & Dose G1

0 mg/kg

b. wt./day G2

10 mg/kg

b. wt. /day G3

100 mg/kg

b. wt./day G4

1000 mg/kg

b. wt./day

Pup Sex M F M F M F M F

N° 52 61 70 87 48 56 63 56

GROSS FINDINGS

EXTERNAL EXAMINATION

No Abnormality Detected 49 64 69 87 47 55 63 56

E maciation 0 0 0 1 1 0 0 0

INTERNAL EXAMINATION

Lungs

Congestion 1 2 1 1 0 2 1 0

Liver

Congestion 1 1 0 1 0 1 0 0

Kidneys

Pallor 0 0 0 1 0 0 0 0

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 151 of 217

APPENDIX 16 (Continued)

APPENDIX A : Terminal Body Weight (g), Absolute Organ Weight (g) and Organ Weight Relative

to Terminal Body Weight (%) - Parental Rat

Group: G1 (Control); Dose: 0 mg /kg b. wt. /day Sex: M ale

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

body weight (%)

Testes Epididymis Testes Epididymis

1 446.8 3.548 1.515 0.794 0.339

2 511.8 4.213 1.600 0.823 0.313

3 412.7 3.433 1.353 0.832 0.328

4 409.6 3.588 1.320 0.876 0.322

5 435.8 4.237 1.723 0.972 0.395

6 373.7 3.772 1.564 1.009 0.419

7 397.8 4.306 1.598 1.082 0.402

8 438.7 4.230 1.482 0.964 0.338

9 422.5 3.841 1.673 0.909 0.396

10 418.3 4.677 1.628 1.118 0.389

11 460.2 4.377 1.536 0.951 0.334

12 521.9 4.252 1.450 0.815 0.278

13 470.2 3.871 1.598 0.823 0.340

14 499.6 3.948 1.640 0.790 0.328

15 500.3 4.234 1.625 0.846 0.325

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 152 of 217

APPENDIX 16 (Continued)

APPENDIX A (Continued)

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

Sex: M ale

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

bod y weight (%)

Testes Epididymis Testes Epididymis

31 404.5 4.242 1.378 1.049 0.341

32 389.4 3.348 1.214 0.860 0.312

33 439.8 4.318 1.369 0.982 0.311

34 414.5 3.751 1.311 0.905 0.316

35 435.8 4.360 1.514 1.000 0.347

36 416.8 3.629 1.245 0.871 0.299

37 451.9 4.268 1.466 0.944 0.324

38 435.2 3.744 1.473 0.860 0.338

39 427.9 3.830 1.659 0.895 0.388

40 437.6 4.093 1.563 0.935 0.357

41 463.1 4.240 1.621 0.916 0.350

42 379.7 3.960 1.400 1.043 0.369

43 456.3 3.893 1.532 0.853 0.336

44 393.6 3.449 1.343 0.876 0.341

45 442.7 4.132 1.475 0.933 0.333

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 153 of 217

APPENDIX 16 (Continued)

APPENDIX A (Continued)

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

Sex: M ale

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

body weight (%)

Testes Epididym is Testes Epididymis

61 400.4 3.714 1.250 0.928 0.312

62 445.7 3.802 1.397 0.853 0.313

63 382.3 3.452 1.464 0.903 0.383

64 422.1 4.328 1.498 1.025 0.355

65 362.4 3.400 1.366 0.938 0.377

66 426.4 4.096 1.418 0.961 0.333

67 494.9 4.859 1.638 0.982 0.331

68 405.3 3.634 1.168 0.897 0.288

69 450.8 4.170 1.611 0.925 0.357

70 449.9 3.862 1.495 0.858 0.332

71 422.8 4.161 1.265 0.984 0.299

72 469.8 2.918 1.014 0.621 0.216

73 434.1 3.929 1.445 0.905 0.333

74 406.6 3.935 1.373 0.968 0.338

75 368.9 3.398 1.144 0.921 0.310

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 154 of 217

APPENDIX 16 (Continued)

APPENDIX A (Continued)

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

Sex: M ale

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

body weight (%)

Testes Epididymis Testes Epididymis

91 423.9 3.899 1.405 0.920 0.331

92 388.7 3.871 1.365 0.996 0.351

93 401.3 3.530 1.453 0.880 0.362

94 421.5 3.692 1.295 0.876 0.307

95 416.8 4.157 1.546 0.997 0.371

96 403.6 3.805 1.311 0.943 0.325

97 464.4 3.969 1.404 0.855 0.302

98 416.1 4.442 1.648 1.068 0.396

99 441.8 3.951 1.443 0.894 0.327

100 423.5 4.137 1.531 0.977 0.362

101 446.6 4.481 1.611 1.003 0.361

102 467.9 4.076 1.497 0.871 0.320

103 471.5 4.180 1.360 0.887 0.288

104 398.2 3.415 1.332 0.858 0.335

105 456.2 4.445 1.526 0.974 0.335

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 155 of 217

APPENDIX 16 (Continued)

APPENDIX B : Macroscopic and Microscopic Findings of Individual Rat

- Parental Rat

Group: G1 (Control) ; Dose : 0 mg /kg b. wt. /day Sex: M ale

Rat N° Mode

of

Death Gross Findings

Microscopic Findings

External Internal

1 TS NAD NAD NAD

2 TS NAD NAD T estes (unilateral): Oedema, interstitial, focal (+)

3 TS NAD NAD NAD

4 TS NAD NAD NAD

5 TS NAD NAD NAD

6 TS NAD NAD NAD

7 TS NAD NAD NAD

8 TS NAD NAD NAD

9 TS NAD NAD NAD

10 TS NAD NAD NAD

11 TS NAD NAD Epididymes (unilateral): Infiltration, MNC, interstitial,

focal (+)

12 TS NAD NAD NAD

13 TS NAD NAD NAD

14 TS NAD NAD NAD

15 TS NAD NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 156 of 217

APPENDIX 16 (Continued)

APPENDIX B (Continued)

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

Rat N° Mode

of

Death Gross Findings Microscopic Findings

External Internal

16 TS NAD NAD NAD

17 TS NAD NAD NAD

18 TS NAD NAD NAD

19 TS NAD NAD NAD

20 TS NAD NAD NAD

21 TS NAD NAD NAD

22 TS NAD NAD NAD

23 TS NAD NAD NAD

24 TS NAD NAD NAD

25 TS NAD NAD NAD

26 TS NAD NAD NAD

27 TS NAD NAD NAD

28 TS NAD NAD NAD

29 TS NAD NAD NAD

30 TS NAD NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 157 of 217

APPENDIX 16 (Continued)

APPENDIX B (C ontinued)

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

Rat N° Mode

of

Death Gross Findings

Microscopic Findings

External Internal

31 TS NAD NAD X

32 TS NAD NAD X

33 TS NAD NAD X

34 TS NAD NAD X

35 TS NAD NAD X

36 TS NAD NAD X

37 TS NAD NAD X

38 TS NAD NAD X

39 TS NAD NAD X

40 TS NAD NAD X

41 TS NAD NAD X

42 TS NAD NAD X

43 TS NAD NAD X

44 TS NAD NAD X

45 TS NAD NAD X

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 158 of 217

APPENDIX 16 (Continued)

APPENDIX B (Continued)

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

Rat N° Mode

of

Death Gross Findings

Microscopic Findings

External Internal

46 TS NAD NAD X

47 TS NAD NAD X

48 TS NAD NAD X

49 TS NAD NAD X

50 TS NAD NAD X

51 TS NAD NAD X

52 TS NAD NAD X

53 TS NAD NAD X

54 TS NAD NAD X

55 TS NAD NAD X

56 TS NAD NAD X

57 TS NAD NAD X

58 TS NAD NAD X

59 TS NAD NAD X

60 TS NAD NAD X

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 159 of 217

APPENDIX 16 (Continued)

APPENDIX B (C ontinued)

Group: G3 (Mid Dose); Dose: 1 00 mg/kg b. wt./day Sex: M ale

Rat N° Mode

of

Death Gross Findings

Microscopic Findings

External Internal

61 TS NAD NAD X

62 TS NAD NAD X

63 TS NAD NAD X

64 TS NAD NAD X

65 TS NAD NAD X

66 TS NAD NAD X

67 TS NAD NAD X

68 TS NAD NAD X

69 TS NAD NAD X

70 TS NAD NAD X

71 TS NAD NAD X

72 TS NAD Testes (unilateral,

right): Reduced s ize (+) 

### Testes (unilateral): Degeneration

/exfoliation, germ cells, tubular

, multifocal (1+)

73 TS NAD NAD X

74 TS NAD NAD X

75 TS NAD NAD X

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 160 of 217

APPENDIX 16 (Continued)

APPENDIX B (Continued)

Group: G3 (Mid Dose); Dose: 1 00 mg/kg b. wt./day Sex: Female

Rat N° Mode

of

Death Gro ss Findings

Microscopic Findings

External Internal

76 TS NAD NAD X

77 TS NAD NAD X

78 TS NAD NAD X

79 TS NAD NAD X

80 TS NAD NAD X

81 TS NAD NAD X

82 TS NAD NAD X

83 TS NAD NAD X

84 TS NAD NAD X

85 TS NAD NAD X

86 TS NAD NAD X

87 TS NAD NAD X

88 TS NAD NAD X

89 TS NAD NAD X

90 TS NAD NAD X

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 161 of 217

APPENDIX 16 (Continued)

APPENDIX B (C ontinued)

Group: G 4 ( High Dose); Dose: 10 00 mg/kg b. wt./day Sex: M ale

Rat N° Mode

of

Death Gross Findings

Microscopic Findings

External Internal

91 TS NAD NAD NAD

92 TS NAD NAD Epididymes (unilateral): Infiltration, MNC, interstitial,

focal (+)

93 TS NAD NAD NAD

94 TS NAD NAD Testes (unilateral): Oedema, interstitial, focal (+)

95 TS NAD NAD NAD

96 TS NAD NAD NAD

97 TS NAD NAD NAD

98 TS NAD NAD NAD

99 TS NAD NAD NAD

100 TS NAD NAD NAD

101 TS NAD NAD NAD

102 TS NAD NAD NAD

103 TS NAD Epididymis

(Right,

unilateral):

Nodule, white,

cauda (+) Epididymes (unilateral): Granuloma, sperms,

multifocal (2+); Reduced sperms, tubular lumen,

multifocal (2 +)

104 TS N AD NAD NAD

105 TS NAD NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 162 of 217

APPENDIX 16 (Continued)

APPENDIX B (Continued)

Group: G 4 ( High Dose); Dose: 10 00 mg/kg b. wt./day Sex: Female

Rat N° Mode

of

Death Gross Findings

Microscopic Findings

External Internal

106 TS NAD NAD NAD

107 TS NAD NAD NAD

108 TS NAD NAD NAD

109 TS NAD NAD NAD

110 TS NAD NAD NAD

111 TS NAD NAD NAD

112 TS NAD NAD NAD

113 TS NAD NAD NAD

114 TS NAD NAD NAD

115 TS NAD NAD NAD

116 TS NAD NAD NAD

117 TS NAD NAD NAD

118 TS NAD NAD NAD

119 TS NAD NAD NAD

120 TS NAD NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 163 of 217

APPENDIX 16 (Continued)

APPENDIX C : Gross Findings of Individual F1 Preweanling (4 th 

### Day) Pups

Group: G1 (Control) Dose: 0 mg/kg b. wt./day

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

16 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 F TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

17 1 M TS NAD NAD

2 M TS NAD NAD

3 F TS NAD NAD

4 F TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 164 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

Group: G1 (Control) Dose: 0 mg/kg b. wt./day

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

18 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M FD NAD Lungs: Congestion (+)

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

19 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 M TS NAD NAD

9 M TS NAD NAD

10 M TS NAD NAD

11 M TS NAD NAD

12 M TS NAD NAD

13 M FD NAD Liver: Congestion (+)

14 F TS N AD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 165 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

Group: G1 (Control) Dose: 0 mg/kg b. wt./day

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

20 1 M TS NAD NAD

2 M TS NAD NAD

3 F TS NAD NAD

22 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD N AD

4 F TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F FD NAD Liver: Congestion (+)

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 166 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

Group: G1 (Control); Dose: 0 mg/k g b. wt./day

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

23 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F FD NAD Lungs: Congestion (+)

24 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

15 F TS NAD NAD

16 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 167 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

27 1 M TS NAD NAD

2 M TS N AD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 F TS NAD NAD

29 2 F FD NAD Lungs: Congestion (1+)

30 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 168 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

46 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 F TS NAD NAD

6 F TS NA D NAD

7 F TS Emaciation (1+) NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

47 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 M TS NAD NAD

9 M TS NAD NAD

10 M FD NAD Lungs: Congestion (+)

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 169 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

48 1 M TS NAD NAD

2 M TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

49 3 M TS NAD NAD

4 F TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

50 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NA D NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

15 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 170 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

51 1 M TS NAD NAD

2 F TS NAD NAD

3 F TS NAD NAD

4 F TS NAD NAD

5 F TS NAD NAD

52 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

15 F FD NAD Lungs: Congestion (+)

Liver: Congestion (1+)

Kidney: Pallor (1+)

16 F TS NAD N AD

53 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

15 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 171 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

54 1 M TS NAD NAD

2 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 F TS NAD N AD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

55 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

15 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 172 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

57 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

58 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 173 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

59 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

60 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 174 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

Group: G3 (Mid Dose); Dose: 1 00 mg/kg b. wt./day

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Finding s

External Internal

78 1 M TS NAD NAD

3 F TS NAD NAD

4 F TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

79 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS Emacia tion (1+) NAD

8 M TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

81 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 F TS NAD NAD

5 F TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 175 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

Group: G3 (Mid Dose); Dose: 1 00 mg/kg b. wt./day

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

82 1 F TS NAD NAD

2 F TS N AD NAD

3 F TS NAD NAD

4 F FD NAD Lungs: Congestion (+)

84 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 M TS NAD NAD

9 M TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

15 F TS NAD NAD

16 F TS NAD NAD

85 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 176 of 217

APPENDIX 16 (Continued)

APPENDIX C: (C ontinued)

Group: G3 (Mid Dose); Dose: 1 00 mg/kg b. wt./day

Dam

N o 

### Pup N o 

### Sex Mode of Death Gross Findings

External Internal

86 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

88 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 F TS NAD NAD

5 F TS NAD NAD

89 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD N AD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 M TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 177 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of

Death Gross Findings

External Internal

90 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 F TS NAD NAD

8 F FD NAD Lungs: Congestion (+)

Liver: Congestion (+)

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 178 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of

Death Gross Findings

External Internal

107 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

108 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 179 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of

Death Gross Findings

External Internal

109 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

111 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 F T S NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 180 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of

Death Gross Findings

External Internal

112 2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 M FD NAD Lungs: Congestion (+)

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

14 F TS NAD NAD

15 F TS NAD NAD

115 2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 181 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of

Death Gross Findings

External Internal

116 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 M TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

117 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS N AD NAD

13 F TS NAD NAD

### JRF Study Number: 496 -1-04- 4306 ( F inal Report) Page 182 of 217

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

N o 

### Pup N o 

### Sex Mode of

Death Gross Findings

External Internal

118 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 F TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

119 1 M TS NAD NAD

2 M TS NAD NAD

3 M TS NAD NAD

4 M TS NAD NAD

5 M TS NAD NAD

6 M TS NAD NAD

7 M TS NAD NAD

8 M TS NAD NAD

9 F TS NAD NAD

10 F TS NAD NAD

11 F TS NAD NAD

12 F TS NAD NAD

13 F TS NAD NAD

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 183 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 1 7

Analytical Report on Residues of Organochlorine and Organophosphorus Pesticides and Heavy

Metals in F oo d, Water and Rice Husk

A. Chemical Analysis Report of Rodent Food

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 184 of 217

APPENDIX 1 7 (Continued)

B. Chemical Analysis Report of Rice Husk

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 185 of 217

APPENDIX 1 7 (Continued)

C . Chemical Analysis Report of Water

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 186 of 217



### Reproduction/Developmental O ral Toxicity Screening Test of

Sodium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 18

Results of Microbiological Analysis of Rat Pellet F oo d, Water and Rice Husk

A. Microbial Analysis Report of Rat Pellet F ood

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 187 of 217

APPENDIX 18 (Continued)

B. Microbial Analysis Report of Water

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 188 of 217

APPENDIX 18 (Continued)

C. Microbial Analysis Report of Water

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 189 of 217

APPENDIX 18 (Continued)

D . Microbial Analysis Report of Rice Husk

### JRF Study Number: 496-1-04-4306 ( Final Report) Page 190 of 217



### Reproduction/Developmental Oral Toxicity Screening Test of

Sod ium 3- nitrobenzene Sulfonate (CAS # 127-68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 1 9

Nutrient Analysis Report of Rat Pellet F ood

### JRF Study Number: 496-1-04- 4306 (Final Report) Page 191 of 21 7



### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3

- nitrobenzene Sulfonate (CAS # 127 -68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 20

Signed Study Plan and Study Plan Amendment s

JRF Study Number: 496 -1- 04

- 4306 Page 1 of 17

STUDY PL AN

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 TIARC O DRIVE

DALTON, 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 ( Final Report) Page 192 of 217

APPENDIX 20 (Continued)

JRF Study Number: 496 -1- 04

- 4306 Page 2 of 17

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 an y) 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 Sani tation

.............................................................................................................. 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

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

### JRF Study Number: 496-1-04- 4306 ( Final Report) Page 193 of 217

APPENDIX 20 (Continued)

JRF Study Number: 496 -1- 04

- 4306 Page 3 of 17

CONTENTS (Continued)

PAGE N°

6. EX PERIMENTAL PROCEDURE

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

6.1 Vehicle and Justification for Choice

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

6.2 Route and Mode of Administration and Justification for Choice

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

6.3 Dose Levels and Justification

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

6.4 Preparation of Dose Formulations

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

6.5 Stability of the Test Item in the Vehi cle

.............................................................................................. 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 Organs/Tissues

............................................................................................................ 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 ( Final Report) Page 194 of 217

APPENDIX 20 (Continued)

JRF Study Number: 496 -1- 04

- 4306 Page 4 of 17

1. GENERAL INFORMATION

1.1 Study Professionals

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.

Assist ant Director, Department of Toxicology,

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 : Jeff Vanderlip

706/277

- 1300

Tiarco Chemical

1300 Tiarco Drive

Dalton, Georgia 30720

1.4 Regulatory Contact : Bennett King

864/457

- 2321

Tiarco Chemical

1300 Tiarco Drive

Dalton, Georgia 30720 Name of P ersonnel Qualification Profession Experience Training

Deepak Ujawane

(Study Director) M. Pharma. Toxicologist 3 years Declared Study Director for all

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 Toxico

pathologiest

Sanjay Dived ia M.Sc. Chemist 10 years Trained for dose formulation

analysis

### JRF Study Number: 496-1-04- 4306 ( Final Report) Page 195 of 217

APPENDIX 20 (Continued)

JRF Study Number: 496 -1- 04

- 4306 Page 5 of 17

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 S ubmission : 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 of the sodium

3- nitrobenzene sulfonate (CAS No. 127

- 68

- 4) on reproduction and/or d evelopment, 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. “Reproduction/Developmental Toxicity Screening Test”

.

2.3 Test Method Principle

The sodium 3

- nitrobenzene sulfonate (CAS No. 127

- 68

- 4) will be administered in gra duated 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 be fore 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 ( Final Report) Page 196 of 217

APPENDIX 20 (Continued)

JRF Study Number: 496 -1- 04

- 4306 Page 6 of 17

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

toxicity. Animals, whic h 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 r epresentative 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

IUPAC Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CA Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CAS N° : 127

- 68 -4

Batch N° : 50623

Concentration / Puri ty : 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 fro m 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 °F in the Test Item Control Office

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

of the Spons or.

JRF Test Item Code : SNB 444

### JRF Study Number: 496-1-04- 4306 ( Final Report) Page 197 of 217

APPENDIX 20 (Continued)

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), ENV /MC/CHEM (98)17, N° 1, 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 Stan dard

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 Direc tor 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

wi ll 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 Q uality 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 o f 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 rele vant to humans and animals.

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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 cons istent 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 Humane 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 SY STEM

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 will be

nulliparous and non

- pregnant. At the initiation of the acclimatization rats will of 10 to 11weeks 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 avail able 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 wil l 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.

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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, lig ht 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 of 2 rats/cage (one male plus one female). Mated female rat will be caged

individually and nesting material (shredded paper) at near term or around 14 th 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 ric e (paddy) husk as the bedding.

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

analysis of samples of the bedding material will be performed at six months intervals at Jai Research

Foundation and mo st 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 o

JRF/SSI/SOP

- 829) sex

- wise. Prior to the start of the t reatment, 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.

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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/TOX/SOP

- 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./day), Animal N°.

5.11 Mating Procedure

During the mating period, the male and female rats will be mated in a 1:1 mating ratio to procure the F 1

generatio n. 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, mate d female rats will be caged individually.

For each mating, each female will be placed with a single male from the same dose level (1:1mating) until

copulation occurs or 2 weeks have elapsed. If mating has not occurred during two weeks mating period, re

m ating 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 Administration and Justification for Choice

The route of Sodium 3

- nitrobenzene sulfonate (CAS No. 127

- 68

- 4) administra tion 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 t he test item

and will be intimated to the Sponsor through a formal study plan amendment.

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6.4 Preparation of Dose Formulations

Required quantity of test item for each dose group will be received from TICO. T est 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 of the study by using

validated analytical method

.

6.6 Homogeneity and Active In gredient 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 st udy.

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 mL/kg 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 Experimental Schedule

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

- partu m day 3.

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

Group

N° Dose

(mg/kg b. wt./day) Total N° of Rat Minimum N° of Pregnant Female Rat

Require for Study Male Female

G1 Control (0) 15 15 10

G2 Low Dose 15 15 10

G3 Mid Dose 15 15 10

G4 High Dose 15 15 10

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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 terminally 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 Weigh ts

Individual 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 lactatio n periods food consumption will be measured weekly at a

minimum. The feed consumption will be determined 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

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

be performed using indelible ink.

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7.3 Necropsy

7.3.1 All the survived rat rats will be sacrificed by using over dose of CO 2 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 Weight

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

7.5 Preservation of Organs/Tissues

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 His topathology

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 spermatoge nesis 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 typ es, 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 org ans.

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8. DATA RECORDING AND EVALUATION

8.1 Data Recording

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

Additionally, all data will be summarized in t abular form.

The findings of this toxicity study will be evaluated in terms 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 gr oss lesions in the identified target organs, infertility, clinical abnormalities,

affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic

effects.

8.2 Data Evaluation

The findings of this toxicity study w ill be evaluated in terms 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, infert ility, clinical abnormalities,

affected reproductive and litter performance, 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

a nd 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 ot herwise 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 information:

1. Test Item:

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

b. Id entification 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.

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APPENDIX 20 (Continued)

JRF Study Number: 496 -1- 04

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4. Test conditions:

a. Rationale for dose level selection.

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

preparation.

c. Details of the administration of the test item.

d. Details of food 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 wei ght/body 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 of the 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 of ten 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.

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APPENDIX 20 (Continued)

JRF Study Number: 496 -1- 04

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11. REFERENCES

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

(HPV 3).

40 CFR 799.9355 TSCA Reproduction/Developmental Toxicity Screening Test.

Fisher, R.A. and Yates, F. (1963): Statistical Tables for Biological, A gricultural and Medical Research. 6 th

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,

3 rd Edition, Hayes A.W. (Ed), Raven press Ltd., New York, p. 2 21

- 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– 00

– 367, July 2000. “Reproduction/Developmental Toxicity Screening Test".

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APPENDIX 20 (Continued)

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APPENDIX 20 (Continued)

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APPENDIX 20 (Continued)

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APPENDIX 20 (Continued)

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APPENDIX 20 (Continued)

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APPENDIX 20 (Continued)

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APPENDIX 20 (Continued)

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APPENDIX 20 (Continued)

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### Reproduction/Developmental Oral Toxicity Screening Test of

Sodium 3

- nitrobenzene Sulfonate (CAS # 127 -68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 22

Certificate of Analysis of Sodium 3

- nitrobenzene Sulfonate

### JRF Study Number: 496-1-04- 4306 ( Final Report) Page 217 of 217



### Reproduction/Developmental Oral Toxici ty Screening Test of

Sodium 3

- nitrobenzene Sulfonate (CAS # 127 -68-4; US EPA HPV 3) in Wistar Rats

APPENDIX 23

GLP Endorsement of Compliance