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 

STATEMENT 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 Guidelines ................................................................................................... 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 Instruments 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 Experimental 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 Mating Procedure........................................................................................................... 17 

6. EXPERIMAENTAL PROCEDURE ..................................................................................... 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.......................................................................................... 18 
JRF 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 Homogeneity 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 Preservation of Organs/Tissues...................................................................................... 20 

7.6 Histopathology............................................................................................................... 20 

8. INDICES CALCULATION................................................................................................... 21 

9. DATA RECORDING AND EVALUTION........................................................................... 22 

9.1 Data Recording .............................................................................................................. 22 

9.2 Data Evaluation.............................................................................................................. 22 

10. RESULTS AND DISCUSSION ............................................................................................ 23 

10.1 Parental Data.................................................................................................................. 23 

 10.1.1 Mortality ........................................................................................................... 23 

 10.1.2 Clinical 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 Index........................................................................... 24 

 10.2.5 Litter Size and Sex Ratio .................................................................................. 24 

10.3 Homogeneity 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 Macroscopic Findings....................................................................................... 25 

 10.4.3 Microscopic Findings ....................................................................................... 25 

11. CONCLUSION...................................................................................................................... 26 

12. REFERENCES....................................................................................................................... 27 
JRF 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 – Female (P).............................................................. 30 

4. Summary 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 Food Consumption (g) – Male (P)........................................... 35 

9. Summary of Cage wise Food 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 Symptoms 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. Weekly Body Weight Change (%) of Individual Female Rat (P).................................. 71 

7. Food Consumption (g) of Individual Male Rat (P)........................................................ 75 

8. Food Consumption (g) of Individual Female Rat (P) .................................................... 79

9. Pre-coital Interval and Duration of Gestation Days 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 ...................................... 100 

12. Body Weight (g) of Individual Pups ............................................................................ 104 

13. Mean Pup’s Body Weight (g) of Individual Female.................................................... 117 

14. Pre and Postnatal Data ................................................................................................. 121 

15. Dose Formulation Analysis Report.............................................................................. 125 

16. Pathology Report ......................................................................................................... 141 

17. Analytical Report on Residues of Organochloride and Organophosphorus 

pesticides and Heavy Metals in Food, Water and Paddy Husk.................................... 183 

18. Result of Microbiological Analysis of Rat Pellet Food, Water and Paddy Husk ........ 186 

19. Nutrient Analysis Report of Rat Pellet Food ............................................................... 190 

20. Signed Study Plan and Study Plan Amendments ......................................................... 191 

21. Record of Deviations from Study Plan ........................................................................ 215 

22. Certificate of Analysis of sodium 3-nitrobenzene sulfonate........................................ 216 

 23. GLP Endorsement of Compliance ............................................................................... 217 

 

JRF Study Number: 496-1-04-4306 (Final Report) Page 9 of 217

LIST OF ABBREVIATIONS

BMR - Barrier Maintained Rodent 

b. wt. - Body Weight

F - Female

g - Gram

G - Group

G1 - Control (Reverse Osmosis water) 

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

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

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

JRF - Jai Research Foundation

 - - Not Applicable

M - Male

mL - Millilitre

mg - Milligram

NA - Not Applicable

N - Number of Animals / Observations

N° - Number

n - Number of Cages

NAD - No Abnormalities Detected

OECD - Organisation for Economic Co-operation and Development

P - Parental Animals

PT - Pre-exposure/Pre-treatment

SD - Standard Deviation

TOX - Department of Toxicology

TS - Terminal Sacrifice

TICO - Test Item Control Office

TIDS - Test Item Data Sheet

% - Percent

°C - Degree Centigrade

Note: Sodium 3-nitrobenzene sulfonate (CAS # 127-68-4; US EPA HPV 3) is represented as Sodium 

3-nitrobenzene sulfonate
JRF Study Number: 496-1-04-4306 (Final Report) Page 10 of 217

SUMMARY

Methods

The 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 post-partum day 

3. The methods followed were based on the guidelines 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 doses tested were 10 (G2-Low dose), 100 (G3-Mid dose) 

and 1000 (G4-High dose) mg/kg b. wt./day. A concurrent control group of rats received vehicle only 

(Reverse Osmosis water). 

All rats were observed twice a day for clinical signs and mortality and morbidity 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 days 0 and 4.

At 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 study 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 was observed in parental 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 sulfonate in reverse osmosis was homogeneous and the 

concentration was within the acceptance level.

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

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

index in all the treatment groups was comparable with concurrent control group. No treatment related preimplantation loss, post-implantation loss and post-natal loss was observed in any of the groups treated 

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 1000 

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 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 Guidelines 

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” 

and

40 CFR 799.9355, December 15, 2000. “TSCA Reproduction/Developmental 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 water only. Male rats were dosed for two weeks prior to mating, during mating and up to 

one day after terminal sacrifice of all the females. Female rats were dosed for two weeks 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 : December 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 21, 2013 to January 27, 2013

Experiment Completion : April 19, 2013

 Study Completion : June 14, 2013
JRF 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 amendments, 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° 22) 

Manufactured by : Tiarco Chemical

Supplied by : Reservol 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 item, 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 characterization of the test item were documented in a Certificate of Analysis 

provided by the Sponsor and are included as an appendix (Appendix N° 22) in the study report. 

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

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

responsibility of the Sponsor.

3. MATERIAL

3.1 Instruments and Equipment

Tattoo Machine : Animal Identification & Marking System, Inc. 

Balances : 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 Cannula : 5.0 mL Borosilicate Glass Syringes, 16 gauze x 5 cm rat 

cannula 

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 : Reverse Osmosis Water

 

4. ANIMAL WELFARE

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

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

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

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

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

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

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

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

welfare. 

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

Committee (IAEC), Jai Research Foundation. 

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

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

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

5.2 Justification 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 testing and recommended by various regulatory authorities. In addition, a 

large amount of background knowledge was accumulated on this strain.
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5.3 Location of the Study

During the study period, the test animals were located in the Barrier Maintained Rodent Facility 

(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 this report.

5.5 Acclimatization

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

the experimental procedure room and acclimatized for a period of 7 days prior to randomization. 

For randomization 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 Maintained 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 given in below table:

Environmental 

Parameters

Temperature (°C) Relative 

Humidity (%) Air Change

(per hour)

Mean Light 

Intensity 

Maximum Minimum Maximum Minimum (LUX)

December 2012 23 20 67 65 20 189

January 2013 23 20 66 58 19 220

5.7 Housing

Throughout 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 (shredded paper) from 

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

(size: 41.0 cm x 28.2 cm x 18.0 cm). Each cage was fitted with a stainless steel top grill having 

provision for keeping rat pellet food and a polypropylene 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 inside the Barrier Maintained Rodent Facility till used. The bottom of the cages was 

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

pre-mating period, once in a week.
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Paddy husk is being routinely analyzed once in six months interval for commonly found 

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

18).

5.8 Diet and Water

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

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

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

autoclave sterilised polypropylene bottles. 

Quality of 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 

Appendices 17, 18 and 19). 

5.9 Grouping

Prior to randomization, the rats were weighed. Healthy rats were allocated to the study. Rats were 

randomly allocated to different groups using an in-house developed validated computer software 

program (Gad and Weil, 1994). Animals were divided into four groups [G1 (control), G2 (low 

dose), G3 (mid dose), 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 Identification

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

pen on the dorsal surface of the tail. After grouping, individual 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, the 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 

observed 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 mating pairs was maintained in the raw data. 

Care was taken to avoid mating of siblings.
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6. EXPERIMENTAL PROCEDURE

6.1 Vehicle and Justification for Choice

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

JRF and after 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 Justification 

 Dose levels were selected after consultation with Sponsor based on dose range finding study “JRF 

Study N° 410-1-04-5132”. In this study, three dose levels i.e., 10, 100 and 1000 mg/kg b. wt./day 

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

control group.

Based on this dose levels selected were 0 (G1-Control), 10 (G2-Low), 100 (G3-Mid) and 1000 

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

6.4 Preparation of Dose Formulations

Test item was dissolved in RO Water to obtain desired concentrations. The test formulations were 

prepared every day prior to dosing and used within 2 hours. Prior to dosing, test 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 of dose formulation analysis is appended in Appendix 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 recently recorded body weight. The control group animals were 

given vehicle only. First day of dosing were designated as day 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 (one 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. Pup, which died during study, were 

weighed and subjected to post-mortem examination.

7.1.2 Clinical 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 male 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 was 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 was 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. Pups found dead on the day of littering were examined for possible 

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). Individual pup body weight was recorded on lactation days 0 and 4. 

7.3 Necropsy

All the surviving rats were sacrificed by using over dose of CO2 and live pups were sacrificed by 

intraperitoneal injection of sodium thiopentone (barbiturates). Male rats were sacrificed one day 

after terminal sacrifice of all the females. Female rat along with the pups were sacrificed on postpartum 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 death 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 recorded in dams. 

7.4 Organ Weight

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

7.5 Preservation of Organs/Tissues

At the time of sacrifice, following organs/tissues of rats were 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, testes and epididymis (preserved in Bouin’s fixative)

Organs with gross lesion (preserved in 10% neutral buffered formalin)

7.6 Histopathology

Detailed histological examination was 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 3-6μm thickness) were conducted with special emphasis on 

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

should identify 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 includes the caput, corpus and cauda, which can be accomplished by 

evaluation 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. Fertility index:

5. Gestation index:

6. Live birth index:

7. Viability index on day 4 post-partum:

100

Number of implantations

Number of implantation sites - Number of live concepti 

100

Number of paired animals

Number of mated animals

100

Number of corpora lutea

Number of corpora lutea - Number of implantation sites

100

Number of pregnant females

Number of females with live pupsborn 

100

Number of delivered pups 

Number of live pupsborn 

100

Number of live pupsborn 

Number on pups surviving of day -post 4 partum

100

Number of mated pairs

Number of pregnant female partners
JRF 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. 

Additionally, 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. Statistical analyses on pup data (e.g., number of pups 

born, pup body weights, pup survival) were conducted on a per-litter basis.

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

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

test, Student's t-test, Chi-square test and test of significance for difference of proportions. 

Significance between control and treatment groups 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 Mortality (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 (Refer Tables 2 and 3; Appendices 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 Weight 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, statistically 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 

increase 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 levels showed inconsistent decrease in percent body weight 

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

dependency 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 Tables 8 and 9; Appendices 7 and 8) 

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

compared to concurrent control group.

10.1.5 Fertility Data (Refer Table 12; Appendices 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 postnatal loss in sodium 3-nitrobenzene sulfonate treated groups as compared to control group.

10.2 Pup Data

10.2.1 Mortality (Refer Table 12)

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-nitrobenzene sulfonate treated groups

when compared with control group. 

10.2.2 Clinical Sign (Refer Appendix 10)

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

weakness) were observed in pups belonging to all groups.

10.2.3 Body Weight (Refer Table 11; Appendices 12 and 13)

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; Appendix 11)

Mean male, female and 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 1000 mg/kg b. wt./day dose levels, respectively. The 

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

98.04 and 97.04% at 10, 100 and 1000 mg/kg b. wt./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-nitrobenzene 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 16)

10.4.1 Organ Weight

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

epididymis) 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 pathological 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 observed 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 development up to the 

dose level of 1000 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 Reproduction/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. 6th Edition, Oliver and Boyd, London, U.K., p. 146.

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

Toxicology, 3rd Edition, Hayes A.W. (Ed), Raven press Ltd., New York, pp. 221-274.

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

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

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

The Organization for Economic Co-operation and Development (OECD) Guidelines for the 

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

Screening Test”, adopted 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 25th 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 & 120 

(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) 14 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 Day 
JRF 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°

Observation 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 48
JRF Study Number: 496-1-04-4306 (Final Report) Page 44 of 217 

APPENDIX 1 (Continued) 

Group : G2 Dose : 10 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 Normal 1 48 48

44 Normal 1 49 49 Normal 1 48 48

45 Normal 1 49 49 Normal 1 48 48
JRF Study Number: 496-1-04-4306 (Final Report) Page 45 of 217 

APPENDIX 1 (Continued) 

Group : G3 Dose : 100 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

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 Normal 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 Normal 1 48 48

75 Normal 1 49 49 Normal 1 48 48
JRF 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

Total 

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 Normal 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 Normal 1 48 48
JRF 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 Normal 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 18
JRF 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°

Observation 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 Normal 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 22
JRF 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 Normal 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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 50 of 217 

APPENDIX 2 (Continued)

Group : G2 Dose : 10 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

46 Normal 1 16 16 Normal 1 15 15

47 Normal 1 19 19 Normal 1 18 18

48 Normal 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 16
JRF 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°

Observation 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 Normal 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 23
JRF 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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 53 of 217 

APPENDIX 2 (Continued)

Group : G3 Dose : 100 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 15
JRF Study Number: 496-1-04-4306 (Final Report) Page 54 of 217 

APPENDIX 2 (Continued)

Group : G3 Dose : 100 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 22
JRF Study Number: 496-1-04-4306 (Final Report) Page 55 of 217 

APPENDIX 2 (Continued)

Group : G3 Dose : 100 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-pregnant
JRF 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 Normal 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 15
JRF 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 Normal 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 25
JRF Study Number: 496-1-04-4306 (Final Report) Page 58 of 217 

APPENDIX 2 (Continued)

Group : G4 Dose : 1000 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

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-pregnant
JRF Study 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°

Treatment 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

15 301.8 336.1 369.3 396.1 427.6 455.7 479.8
JRF Study Number: 496-1-04-4306 (Final Report) Page 60 of 217 

APPENDIX 3 (Continued)

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

45 339.8 368.4 389.3 402.6 412.6 426.6 438.1
JRF Study Number: 496-1-04-4306 (Final Report) Page 61 of 217 

APPENDIX 3 (Continued)

Group: G3 Dose: 100 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

70 325.6 356.1 380.5 391.7 407.5 423.8 438.7

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

75 294.7 309.0 323.6 330.8 338.1 351.4 362.4
JRF Study Number: 496-1-04-4306 (Final Report) Page 62 of 217 

APPENDIX 3 (Continued)

Group: G4 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

93 289.1 313.0 336.0 352.1 371.6 385.2 394.0

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

105 328.1 365.0 388.5 403.0 419.8 436.7 452.2
JRF Study 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 - 265.2 281.8 315.5 308.7 311.7 - - 

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 were non-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 64 of 217 

APPENDIX 4 (Continued)

Group: G2 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

54 225.4 239.7 247.9 - 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 was non-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 65 of 217 

APPENDIX 4 (Continued)

Group: G3 Dose: 100 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 - 238.4 256.8 280.5 321.2 - 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 - 258.5 290.8 326.8 390.6 - 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 were non-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 66 of 217 

APPENDIX 4 (Continued)

Group: G4 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 - 292.1 311.1 317.2 375.9 - 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-pregnant
JRF 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 58.98
JRF 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 2.51 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 28.93
JRF Study Number: 496-1-04-4306 (Final Report) Page 69 of 217

APPENDIX 5 (Continued)

Group : G3 Dose : 100 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 22.97
JRF 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 37.82
JRF 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 18.17 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-pregnant
JRF 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 21.76 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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 73 of 217

APPENDIX 6 (Continued)

Group : G3 Dose : 100 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 9.35 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 were non-pregnant
JRF 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 12.74 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-pregnant
JRF 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

Food Consumption (g) of Individual Male Rat (P)

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

Rat N°

During Treatment 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 Sacrifice
JRF 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 20.7 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 Sacrifice 
JRF Study Number: 496-1-04-4306 (Final Report) Page 77 of 217 

APPENDIX 7 (Continued)

Group: G3 Dose: 100 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 Sacrifice
JRF 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 Sacrifice
JRF 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

Food Consumption (g) 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 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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 80 of 217 

APPENDIX 8 (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 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 20.2 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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 81 of 217 

APPENDIX 8 (Continued)

Group: G3 Dose: 100 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 

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-pregnant
JRF 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 Period 

(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-pregnant
JRF 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-pregnant
JRF 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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 85 of 217 

APPENDIX 9 (Continued)

Group: G3 Dose: 100 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-pregnant
JRF 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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 87 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 10

Clinical Symptoms of Individual Pups 

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

Rat N° Pup N° Sex Lactation 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 = Dead
JRF Study Number: 496-1-04-4306 (Final Report) Page 88 of 217 

APPENDIX 10 (Continued)

Group : G1 Dose : 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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 89 of 217 

APPENDIX 10 (Continued)

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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 90 of 217 

APPENDIX 10 (Continued)

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 = Weakness
JRF Study Number: 496-1-04-4306 (Final Report) Page 91 of 217 

APPENDIX 10 (Continued)

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 = Dead
JRF Study Number: 496-1-04-4306 (Final Report) Page 92 of 217 

APPENDIX 10 (Continued)

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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 93 of 217 

APPENDIX 10 (Continued)

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 = Normal
JRF Study Number: 496-1-04-4306 (Final Report) Page 94 of 217 

APPENDIX 10 (Continued)

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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 95 of 217 

APPENDIX 10 (Continued)

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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 96 of 217 

APPENDIX 10 (Continued)

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 = Dead
JRF Study Number: 496-1-04-4306 (Final Report) Page 97 of 217 

APPENDIX 10 (Continued)

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

Rat N° Pup N° Sex Lactation Day

0 1 2 3 4

107 

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 applicable, 1 = Normal, C = Cannibalised

Note: Rat N° 106 and 110 were non-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 98 of 217 

APPENDIX 10 (Continued)

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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 99 of 217 

APPENDIX 10 (Continued)

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

Note: Rat N° 120 was non-pregnant 
JRF 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

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)

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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 101 of 217 

APPENDIX 11 (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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 102 of 217 

APPENDIX 11 (Continued)

Group : G3 Dose : 100 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)

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 11 (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 Screening Test of 

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

APPENDIX 12 

Body Weight (g) of Individual Pups 

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 7.4
JRF Study Number: 496-1-04-4306 (Final Report) Page 105 of 217 

APPENDIX 12 (Continued)

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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 106 of 217 

APPENDIX 12 (Continued)

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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 107 of 217 

APPENDIX 12 (Continued)

Group : G2 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 applicable
JRF Study Number: 496-1-04-4306 (Final Report) Page 108 of 217 

APPENDIX 12 (Continued)

Group : G2 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 applicable
JRF Study Number: 496-1-04-4306 (Final Report) Page 109 of 217 

APPENDIX 12 (Continued)

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

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

Note: Rat N° 56 was non-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 110 of 217 

APPENDIX 12 (Continued)

Group : G2 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 applicable
JRF Study Number: 496-1-04-4306 (Final Report) Page 111 of 217 

APPENDIX 12 (Continued)

Group : G3 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 applicable

Note: Rat N° 76, 77 and 80 were non-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 112 of 217 

APPENDIX 12 (Continued)

Group : G3 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 = Female, - = Not applicable

Note: Rat N° 83 was non-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 113 of 217 

APPENDIX 12 (Continued)

Group : G3 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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 114 of 217 

APPENDIX 12 (Continued)

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

Rat 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-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 115 of 217 

APPENDIX 12 (Continued)

Group : G4 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 = Male, F = Female, - = Not applicable

Note: Rat N° 113 and 114 were non-pregnant 
JRF Study Number: 496-1-04-4306 (Final Report) Page 116 of 217 

APPENDIX 12 (Continued)

Group : G4 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-pregnant 
JRF 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 13 

Mean Pups Body weight (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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 118 of 217 

APPENDIX 13 (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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 119 of 217 

APPENDIX 13 (Continued)

Group : G3 Dose : 100 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

Key: M = Male, F = Female, - = Not applicable

Note: Rat N° 76, 77, 80, 83 and 87 were non-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 120 of 217 

APPENDIX 13 (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° 106, 110, 113, 114 and 120 were non-pregnant
JRF 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 14 

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 Implantation

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: Lac. = Lactation, - = Not applicable

Note: Rat N° 21, 25, 26 and 28 were non-pregnant
JRF 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 Implantation

Live 

Pups on

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-pregnant
JRF Study Number: 496-1-04-4306 (Final Report) Page 123 of 217 

APPENDIX 14 (Continued)

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

Rat

N°

Number of Pre-implantation Post-implantation Post-natal

Corpora 

lutea Implantation

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/day

Rat

N°

Number of Pre-implantation Post-implantation Post-natal

Corpora 

lutea Implantation

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 Report
JRF 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

4.4

Mean 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 Sodium 3-Nitrobenzensulfonate Reference Standard Stock Solution 

 A quantity of 103.84 mg Sodium 3-Nitrobenzensulfonate reference standard (98.0 % purity) was

weighed into a volumetric flask of 100 mL capacity, dissolved in 50.0 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 1017.63 mg/L (RS). 

3.2 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard Working Solutions for 

Linear Dynamic Range

Identification

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 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.

3.3 Linear Dynamic Curve 

Figure 1 

 Page 2 of 16
JRF 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

5

Mean 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-Nitrobenzensulfonate reference standard (98.0 % purity) was

weighed into a volumetric flask of 100 mL capacity, dissolved in 50.0 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 1004.50 mg/L (RS1). 

3.5 Preparation of Sodium 3-Nitrobenzensulfonate Reference Standard Working Solutions for 

Linear Dynamic Range 

Identification

of Reference 

Standard

Solution

Solution

Taken

(mL)

Final

Volume

(mL)

Volume Made 

With

Obtained

Concentration

(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 16
JRF 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 flasks 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 concentration (X) was calculated using the following formula:

 Y = b X + a 

 

Y - a 

A.I. concentration (mg/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 purity of test item

 Page 4 of 16
JRF 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 Purity 

of Test Item

(mg/L)

Sodium 3-Nitrobenzensulfonate

Detected 

Concentration

(mg/L)

Mean 

Concentration 

(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 (< LOQ = 0.5 mg/L)

 Purity of Test Item = 96.9 % 

 Page 5 of 16
JRF 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 

Concentration

(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 (< LOQ = 0.5 mg/L)

 Purity of Test Item = 96.9 % 

 Page 6 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 131 of 217

APPENDIX 15 (continued) 

6. Typical Chromatograms

 Page 7 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 132 of 217

APPENDIX 15 (continued) 

 Page 8 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 133 of 217

APPENDIX 15 (continued) 

 Page 9 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 134 of 217

APPENDIX 15 (continued) 

 Page 10 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 135 of 217

APPENDIX 15 (continued) 

 Page 11 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 136 of 217

APPENDIX 15 (continued) 

 Page 12 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 137 of 217

APPENDIX 15 (continued) 

 Page 13 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 138 of 217

APPENDIX 15 (continued) 

 Page 14 of 16
JRF Study Number: 496-1-04-4306 (Final Report) Page 139 of 217

APPENDIX 15 (continued) 

 Page 15 of 16
JRF 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 (Final Report) Page 142 of 217 

APPENDIX 16 (Continued)

PERSONNEL INVOLVED 

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

Study 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.

PARTHA BOSE, B.Com., P.G.D.C.A. 

 

 

 

 

 
JRF Study Number: 496-1-04-4306 (Final 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 (4th Day) Pups ................................................... 146 

2.4 Microscopic Findings Parental Rat ................................................................................ 146 

3. CONCLUSION ............................................................................................................................ 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 Parental Rat ........................... 149 

C. Gross Findings – Summary by Groups F1 Preweanling (4th Day) Pups ......................... 150 

APPENDICES

A. Terminal Body Weight (g), Absolute Organ Weight (g) and Organ Weight Relative to 

 Terminal Body Weight (%) Parental Rat ........................................................................ 151 

 B. Macroscopic and Microscopic Findings of Individual Rat - Parental Rat ...................... 155 

C. Gross Findings of Individual F1 Preweanling (4th Day) Pups ....................................... 163 
JRF Study Number: 496-1-04-4306 (Final 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 Cell

mg - Milligram 

mL - Milliliter

N - Number of Animals

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 (Final Report) Page 145 of 217 

APPENDIX 16 (Continued)

1. EXPERIMENTAL PROCEDURE

At scheduled sacrifice 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 

thiopentone sodium and subjected to gross necropsy under the direct supervision 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. 

Vagina, 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 histopathological examination of the ovaries, testes and epididymis from the control 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 Relative to 

Terminal Body Weight (%) - Parental Rat [Table A and Appendix A] 

The terminal 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 epididymes 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 Rats [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 (Final Report) Page 146 of 217 

APPENDIX 16 (Continued)

Internal 

Internal examination of the terminally sacrificed animals of all the groups did not reveal any lesion 

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 (4th 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 lesion 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 pathological significance. However, the deceased pups revealed various 

lesions viz. congestion in lungs (G1M: 01/49, G1F: 02/64, G2M: 01/69, G2F: 01/87, G3F: 02/56, 

G4M: 1/63), congestion in liver (G1M: 01/49, G1F: 01/64, G2F: 01/87, G3F: 01/56) and pallor in 

kidneys (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 epididymides 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 portion. 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 significance.

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/exfoliation) and Epididymides (sperm granuloma, reduced 

sperms 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 group (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 (Final Report) Page 147 of 217 

APPENDIX 16 (Continued)

Hence, these lesions were considered to be spontaneous or incidental in nature without an 

indication of an effect of treatment with the test item. 

3. CONCLUSION

Thus it is concluded that the test item [Sodium 3-nitrobenzene 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, 2nd 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. 1st

 Edn. Cache River Press, FL, USA.
JRF Study Number: 496-1-04-4306 (Final Report) Page 148 of 217 

APPENDIX 16 (Continued)

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

Terminal Body Weight (%) – Group Mean Values – Parental Rat

Parameters Absolute organ weight (g) Organ weight relative to terminal 

body weight (%)

Groups G1 G2 G3 G4 G1 G2 G3 G4

Dose 

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

No

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 (Final Report) Page 149 of 217 

APPENDIX 16 (Continued)

TABLE B: Gross and Microscopic Findings – Summary by Groups (Parental rats) 

Keys: M = Male, F = Female

Note: i. Organs not included in table did not reveal any gross and histopathological findings.

ii. ‘X’ = Organs not examined as per study plan / sex difference, except where gross lesions 

were recorded. 

Groups & Dose

G1

0 mg/kg

b. wt./day

G2

10 mg/kg

b. wt./day 

G3

100 mg/kg 

b. wt./day

G4

1000 mg/kg 

b. wt./day

Organs & Lesions

Sex M F M F M F M F

N° of

Animals 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

Reduced 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 (Final Report) Page 150 of 217 

APPENDIX 16 (Continued)

TABLE C: Gross Findings – Summary by Groups F1 Preweanling (0th -4th 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

Emaciation 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 (Final 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: Male

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 (Final Report) Page 152 of 217 

APPENDIX 16 (Continued)

APPENDIX A (Continued)

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

Sex: Male

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

body 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 (Final Report) Page 153 of 217 

APPENDIX 16 (Continued)

APPENDIX A (Continued)

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

Sex: Male

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

body weight (%)

Testes Epididymis 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 (Final Report) Page 154 of 217 

APPENDIX 16 (Continued)

APPENDIX A (Continued)

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

Sex: Male

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 (Final 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: Male

Rat N°

Mode

of

Death

Gross Findings

Microscopic Findings

External Internal

1 TS NAD NAD NAD

2 TS NAD NAD Testes (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 (Final 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 (Final Report) Page 157 of 217 

APPENDIX 16 (Continued)

APPENDIX B (Continued)

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 (Final 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 (Final Report) Page 159 of 217 

APPENDIX 16 (Continued)

APPENDIX B (Continued)

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

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 size (+)

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 (Final Report) Page 160 of 217 

APPENDIX 16 (Continued)

APPENDIX B (Continued)

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

Rat N°

Mode

of

Death

Gross 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 (Final Report) Page 161 of 217 

APPENDIX 16 (Continued)

APPENDIX B (Continued)

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

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 NAD NAD NAD

105 TS NAD NAD NAD
JRF Study Number: 496-1-04-4306 (Final Report) Page 162 of 217 

APPENDIX 16 (Continued)

APPENDIX B (Continued)

Group: G4 (High Dose); Dose: 1000 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 (Final Report) Page 163 of 217 

APPENDIX 16 (Continued)

APPENDIX C: Gross Findings of Individual F1 Preweanling (4th Day) Pups

Group: G1 (Control) Dose: 0 mg/kg b. wt./day 

Dam 

No Pup No 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 (Final Report) Page 164 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

Group: G1 (Control) Dose: 0 mg/kg b. wt./day 

Dam 

No Pup No 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 NAD NAD
JRF Study Number: 496-1-04-4306 (Final Report) Page 165 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

Group: G1 (Control) Dose: 0 mg/kg b. wt./day 

Dam 

No Pup No 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 NAD

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 (Final Report) Page 166 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 (Final Report) Page 167 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No Sex Mode of Death Gross Findings

External Internal

27 

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

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 (Final Report) Page 168 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 NAD 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 (Final Report) Page 169 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam No Pup No 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 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

15 F TS NAD NAD
JRF Study Number: 496-1-04-4306 (Final Report) Page 170 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 NAD

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 (Final Report) Page 171 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 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

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 (Final Report) Page 172 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 (Final Report) Page 173 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 (Final Report) Page 174 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No Sex Mode of Death Gross Findings

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 Emaciation (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 (Final Report) Page 175 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No Sex Mode of Death Gross Findings

External Internal

82 

1 F TS NAD NAD

2 F TS NAD 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 (Final Report) Page 176 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 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 177 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam

No Pup No 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 (Final Report) Page 178 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 (Final Report) Page 179 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 TS NAD NAD

6 F TS NAD NAD

7 F TS NAD NAD

8 F TS NAD NAD
JRF Study Number: 496-1-04-4306 (Final Report) Page 180 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 (Final Report) Page 181 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 NAD NAD

13 F TS NAD NAD
JRF Study Number: 496-1-04-4306 (Final Report) Page 182 of 217 

APPENDIX 16 (Continued)

APPENDIX C: (Continued)

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

Dam 

No Pup No 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 17 

Analytical Report on Residues of Organochlorine and Organophosphorus Pesticides and Heavy 

Metals in Food, 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 17 (Continued)

B. Chemical Analysis Report of Rice Husk
JRF Study Number: 496-1-04-4306 (Final Report) Page 185 of 217 

APPENDIX 17 (Continued)

C. Chemical Analysis Report of Water
JRF Study Number: 496-1-04-4306 (Final Report) Page 186 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 18 

Results of Microbiological Analysis of Rat Pellet Food, Water and Rice Husk

A. Microbial Analysis Report of Rat Pellet Food 
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 

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

APPENDIX 19 

Nutrient Analysis Report of Rat Pellet Food 

 
JRF Study Number: 496-1-04-4306 (Final Report) Page 191 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 20 

Signed Study Plan and Study Plan Amendments 

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

STUDY PLAN

REPRODUCTION/ DEVELOPMENTAL TOXICITY SCREENING TEST OF 

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

GUIDELINES: OPPTS 870.3550

TEST: 40 CFR 799.9355 

TEST RULE: 40 CFR 799.5089

SPONSOR

TIARCO CHEMICAL

1300 TIARCO DRIVE

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 any) Distribution.................................................................................. 5 

2. INTRODUCTION ............................................................................................................................................. 5 

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

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

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

2.4 Test Item ................................................................................................................................................ 6 

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

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

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

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

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

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

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

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

5. TEST SYSTEM ................................................................................................................................................. 8 

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

5. 2 Justification of Selection of Test System............................................................................................... 8 

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

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

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

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

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

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

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

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

5.11 Mating Procedure................................................................................................................................. 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. EXPERIMENTAL 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 Vehicle .............................................................................................. 11

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

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

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

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

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

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

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

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

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

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

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

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

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

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

7.5 Preservation of 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.

 Assistant 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 Personnel 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 Toxicopathologiest

Sanjay Divedia 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 Submission : November 1, 2012

Final Report Submission : December 10, 2012

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

1.6 Study Plan and Amendment (if any) Distribution

a. Original copy in Archive and Study Sponsor; 

b. Photocopy to Study Director, QAU and Pathology

2. INTRODUCTION

2.1 Objective

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

3- nitrobenzene sulfonate (CAS No. 127-68-4) on reproduction and/or development, either at an early stage 

of assessing the toxicological properties of chemicals or on chemicals of concern. 

2.2 Regulatory Guidelines

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

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

870.3550, EPA712–C–00–367, July 2000. “Reproduction/Developmental Toxicity Screening Test”. 

2.3 Test Method Principle

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

groups of males and females. Males will be dosed for a minimum of four weeks (this includes a minimum 

of two weeks prior to mating, during the mating period and, approximately, two weeks post-mating) and up 

to and including the day before scheduled sacrifice (One day after terminal sacrifice of all the females). 

Females will be dosed throughout the study (this includes two weeks prior to mating, the variable time to 

conception, the duration of pregnancy and at least four days after delivery up to and including the day 

before scheduled sacrifice (post-partum 3). 
JRF Study Number: 496-1-04-4306 (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, which die or will be sacrificed under moribund condition during the test period, are 

necropsies and at the time of terminal sacrifice all the surviving animals will be sacrificed.

2.4 Test Item

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

will be retained for archive purpose. Any residual test item will be disposed of at JRF after the expiry date, 

unless otherwise instructed by the Sponsor. The details relating to the test item (as supplied by the study

Sponsor) are as follows:

Test Item Name : Sodium 3- nitrobenzene sulfonate

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 / Purity : 96.9%

Manufactured by : Tiarco Chemical

Supplied by : Tiarco Chemical

Date of Manufacture : September 08, 2011

Date of Expiry : September 08, 2013

Appearance : Yellow to tan granular

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

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

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

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

of the Sponsor. 

JRF Test Item Code : 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 Cooperation and Development, Paris (1998).

3.2 Standard Operating Procedures (SOP)

Unless otherwise specified all procedures mentioned in the study plan are subject to detailed Standard 

Operating Procedures of Jai Research Foundation. 

3.3 Amendment to Study Plan

This study plan may be subject to amendment. Amendment to study plan, whether initiated by the Sponsor 

or the Study Director will be generated, authorized by the Study Director and will be sent to the Sponsor 

for approval.

In the event that circumstances dictate immediate action, the nature of these circumstances will be 

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

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

3.4 Deviation from the Study Plan

Any deviation (unplanned change) from the study plan will be documented in study file and reported in the 

study report.

3.5 Quality Assurance

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

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

audits and reporting of findings to the Study Director and the Management will be incorporated in the study 

report. 

4. ANIMAL WELFARE

The study will be carried out in compliance with the ‘Guidelines for Laboratory Animals Facility’ issued 

by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), 

India. These guidelines promote the humane care of animals used in research by providing specifications 

that will enhance animal well being and experimental quality for the advancement of biological knowledge 

that is relevant to humans and animals. 
JRF Study Number: 496-1-04-4306 (Final Report) Page 198 of 217 

APPENDIX 20 (Continued)

JRF Study Number: 496-1-04-4306 Page 8 of 17

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

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

objectives and in accordance with Jai Research Foundations policy on animal welfare.

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

(IAEC), Jai Research Foundation. 

4.1 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 SYSTEM

5.1 Test System Specification 

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

Animal Breeding Facility, Jai Research Foundation and used for the experiment. Female rats 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 available laboratory model and is historically 

shown as acceptable model for developmental and reproductive toxicity testing and recommended by the 

OPPTS and other regulatory authorities. 

5.3 Location of the Study

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

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

5.4 Experimental Room Sanitation

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

disinfectant solution. Detail of disinfectant will be document in the raw data and reported in the final report. 
JRF Study Number: 496-1-04-4306 (Final Report) Page 199 of 217 

APPENDIX 20 (Continued)

JRF Study Number: 496-1-04-4306 Page 9 of 17

5.5 Acclimatization

The animals will be received into the experimental room and acclimatized for a minimum period of 5 days 

prior to the cohabitation. During the acclimatization period, the animals will be observed daily for clinical 

signs. Prior to cohabitation, Animals with any evidence of ill health or poor physical condition will be 

excluded from the study.

5.6 Environmental Conditions

Rats will be maintained in an environment-controlled room at a temperature of 22 ± 3 °C and relative 

humidity of 30 to 70 percent. The photoperiod will be 12 hours light and 12 hours darkness, light hours 

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

5.7 Housing

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

rats will be housed in groups 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 14th day of gestation will be 

provided. During study, the rats will be housed in solid floor polypropylene rat cages (size: 41cm x 28.2 cm 

x 18 cm). Each cage will be fitted with a stainless steel top grill having provision for keeping rat pellet feed 

and a polypropylene water bottle with stainless steel drinking nozzle. The bottom of the cages will be 

layered with clean sterilized rice (paddy) husk as the bedding. 

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

analysis of samples of the bedding material will be performed at six months intervals at Jai Research 

Foundation and most recent results will be included in the final report. 

5.8 Diet and Water

The experimental animals will be fed ad libitum with standard rodent pellet diet with an unlimited supply 

of clean and filtered drinking water (Reverse Osmosis water filter system) in polypropylene bottles.

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

food supplier. Quality of food and water is being monitored regularly at Jai Research Foundation. The most 

recent result of microbial and chemical contaminant analysis will be reported in the final report. 

5.9 Grouping

The rats will be randomly assigned to four groups using a stratified body weight procedure (SOP No

JRF/SSI/SOP - 829) sex-wise. Prior to the start of the treatment, the body weight of the rat will not exceed 

± 20% of the mean body weight in each sex. Rats out of this weight range will be returned to the animal 

breeding facility, JRF or discarded without any further examination.
JRF Study Number: 496-1-04-4306 (Final Report) Page 200 of 217 

APPENDIX 20 (Continued)

JRF Study Number: 496-1-04-4306 Page 10 of 17

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 F1

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

pregnancy will be recorded. ‘Day 0’ of pregnancy is defined as the day on which sperms are observed in 

the vaginal smears of rats. After evidence of copulation, mated female rats will be caged individually. 

For each mating, each female will be placed with a single male from the same dose level (1:1mating) until 

copulation occurs or 2 weeks have elapsed. If mating has not occurred during two weeks mating period, remating 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) administration will be through oral 

gavage since it will provide an exaggerated model of the reasonably foreseeable exposure in Human.

6.3 Dose Levels and Justification

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

and will be intimated to the Sponsor through a formal study plan amendment.
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6.4 Preparation of Dose Formulations

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

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

every day extemporaneously prior to dosing. If require, it will be thoroughly mixed using magnetic stirrer 

or glass rod in order to administer a homogeneous preparation.

6.5 Stability of the Test Item in the Vehicle

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

validated analytical method. 

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

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

No. 127-68-4) will be analyzed before start and once during the treatment period from the main study. 

Details will be reported in the final report.

6.7 Administration of Doses

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

week. The dose volume for the administration will be between 5 to 10 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 continued during the mating 

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

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

pregnancy and up to post-partum day 3. 

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

Group

N°

Dose

(mg/kg b. wt./day)

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

Male Female Require for Study

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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APPENDIX 20 (Continued)

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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 Weights

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 lactation 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 identification will 

be performed using indelible ink.
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APPENDIX 20 (Continued)

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7.3 Necropsy

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

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

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

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

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

reproductive system.

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

7.4 Organ 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 Histopathology

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

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

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

 

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

paraffin embedding and transverse sections of 4-5±m thickness) will be conducted with special emphasis 

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

identified treatment-related effects such as retained spermatids, missing germ cell layers or types, 

multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact 

epididymis will be included the caput, corpus, and cauda, which can be accomplished by evaluation of a 

longitudinal section. The epididymis will be evaluated for leukocyte infiltration, change in prevalence of 

cell types, aberrant cell types, and phagocytosis of sperm. Histopathological examination of the ovary 

should detect qualitative depletion of the primordial follicle population. PAS and hematoxylin staining will 

be used for examination of the male reproductive organs. 
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APPENDIX 20 (Continued)

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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 tabular 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 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.

8.2 Data Evaluation

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 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.

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

and Dunnett’s ‘t’ test, Student's t-test, Chi-square test and Test of significance for difference of proportions. 

Significance between control and treatment groups will be calculated with statistical significance of 95% 

confidence level.

9. REPORT

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

will be issued to the Sponsor. The report will include the following information:

1. Test Item:

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

b. Identification data.

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

3. Test animals:

a. Species/strain used.

b. Number, age and sex of animals.

c. Source, housing conditions, diet, etc.

d. Individual weights of animals at the start of the test.
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APPENDIX 20 (Continued)

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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 weight/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)

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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, Agricultural and Medical Research. 6th

Edition, Oliver and Boyd, London, U.K., p. 146.

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

3rd Edition, Hayes A.W. (Ed), Raven press Ltd., New York, p. 221-274.

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

Number 1, “OECD Principles on Good Laboratory Practice” ENV/MC/CHEM(98)17 (as revised in 1997).

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

870.3550, EPA712–C–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 Toxicity Screening Test of 

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

APPENDIX 23 

GLP Endorsement of Compliance