JRF Study Number: 401-1- 04 -5131 (Final Report) Page 2 of 43



### STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on the basis of its falling

within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C).

Sponsor Tiarco Chemical

1300 Tiarco Drive

Dalton, GA 30720

Signature

__________________________

Sponsor/Submitter

Date

___________________________JRF Study Number: 401-1- 04 -5131 (Final Report) Page 6 of 43



### CONTENTS

PAGE N°

TITLE PAGE

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

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

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

STATEMENT OF GLP COMPLIANCE

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

STATEMENT OF COMPLIANCE WITH GOOD LABORATORY PRACTICE

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

STATEMENT OF QUALITY ASSURANCE

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

CONTENTS

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

PERSONNEL INVOLVED IN THE STUDY

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

SUMMARY

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

1. INTRODUCTION

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

1.1 Study Objective

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

1.2 Study Guidelines

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

1.3 Justification for Selection of the Test System

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

1.4 Test Facility and Study Period

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

1.5 Archives

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

2. EXPERIMENTAL PROCEDURE

................................................................................................ 11

2.1 Test Item

.............................................................................................................................. 11

2.2 Instruments and Equipments

................................................................................................ 11

2.3 Solvents and Chemical

......................................................................................................... 11

2.4 Animals

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

2.5 Acclimatisation

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

2.6 Husbandry Practices

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

2.7 Animal Identification

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

2.8 Feed and Water

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

2.9 Environmental Conditions

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

2.10 Experimental Activities

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

2.11 Dose Preparation

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

2.12 Preparation of Dosing Solution

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

2.13 Main Study

................................ ................................ ................................ .......................... 1 4

2.14 Observations

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

2.15 Pathology

................................ ................................ ................................ ............................. 14JRF Study Number: 401-1- 04 -5131 (Final Report) Page 7 of 43

CONTENTS (Continued)

PAGE N°

3. RESULTS

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

3.1 Mortalities

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

3.2 Clinical Observations

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

3.3 Body Weight

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

3.4 Necropsy (Macroscopic Findings)

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

3.5 Interpretation of Results

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

4. CONCLUSION

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

5. REFERENCES

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

TABLE

1. Test Sequence and Mortalities

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

APPENDICES

1. Individual Clinical Observations and Mortalities

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

2. Body Weight (g) and Percent Body Weight Change of Individual Rat

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

3. Necropsy Findings of Individual Rat

................................................................................... 21

4. Dose Progression

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

5. Classifications

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

6. Signed Study Plan

................................................................................................................ 27

7. Certificate of Analysis of Sodium 3-nitrobenzensulfonate

..................................................... 41

8. GLP Endorsement of Compliance

....................................................................................... 42JRF Study Number: 401-1- 04 -5131 (Final Report) Page 9 of 43



### SUMMARY

This study was performed to determine the acute oral toxicity (LD 50 ) of sodium 3-nitrobenzensulfonate

(sponsored by Tiarco Chemical, GA) in Wistar rats. The method followed was as per the guidelines of OECD

N° 425 (October 2008) and OPPTS 870.1100, EPA (December 2002).

The acute oral toxicity study (Up and Down Procedure) was conducted in 8

- 1 0 week s old female Wist a r rats

and were given a single oral dose of sodium 3

- nitrobenzensulf onate in distilled water . The main test was

conducted with the starting dose of 175 mg sodium 3

- nitrobenzensulfonate /kg body weight. Subsequently,

eight additional female Wistar rats were given doses according to the Up and Down Procedure (Slope N° 4)

.

No mortality and clinical signs observed at each dose level tested throughout the treatment period.

All rats had a normal gain in body weight during the experimental period

.

External and visceral examination of the terminally sacrificed animals did no t reveal any abnormality of

pathological significance

. In absence of any pathological lesion in terminally sacrificed animals, it is concluded

that the test item did not produce any treatment related effect at the dose level tested in present study

.

Under the conditions of this study, the acute oral LD 50 of sodium 3

- nitrobenzensulfonate was found to be

grea t er than 5000 mg/kg body weight in female Wistar rats.

Sodium 3

- nitrobenzensulfonate is being classified as follows:

Globally Harmonized System (GHS 20 11 ) : Category 5

EPA Toxicity Categories (December 2002) : Category IVJRF Study Number: 401-1- 04 -5131 (Final Report) Page 10 of 43



### 1. INTRODUCTION

1.1 Study Objective

This study was performed to assess the acute oral toxicity (LD 50 ) of sodium 3

- nitrobenzensulfonate in

Wistar rats. The study was conducted in compliance with the OECD Principles of GLP (1998).

1.2 Study Guidelines

The present study was conducted according to :

The United States Environmental Protection Agency (December 2002). Health Effects Test

Guidelines, OPPTS 870.1100, EPA 712

- C

- 02

- 190, “ Acut e Oral Toxicity ” ;

and

The Organisation for Economic Co

- operation and Development (OECD) Guidelines for Testing of

Chemicals, N° 425, “Acute Oral Toxicity

– Up

- and

- Down Procedure”, adopted by the Council on

October 03, 2008

.

1.3 Justification for Selection of the Test System

The rat ( Rattus norvegicus ) was selected as the test system because it is a readily available rodent

species. It has been historically shown to be a suitable model for acute oral toxicity assessment and is

recommended by EPA, OECD and other regulatory authorities. The results of the study are expected

to be of value in predicting the acute toxicity of the test item in humans and higher mammals.

1.4 Test Facility and Study Period

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

- 396

108, Dist. Valsad, Gujarat, India.

Study Initiation : August 10 , 201 2

Experiment Start : August 21 , 201 2

Experiment Completion : September 26 , 2012

Study Completion : October 19, 2012

1.5 Archives

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

the signed study plan , the draft report, a copy of the final report and the representative sample of the

test item will be retained in the GLP Archives at Jai Research Found ation 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 fo r the material to be disposed o f.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 11 of 43



### 2. EXPERIMENTAL PROCEDURE

2.1 Tes t Item

Details of the test item provided by the Sponsor (Ref. TIDS) :

Test Item Name : Sodium 3

- nitrobenzensulfonate

IUPAC Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CA Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CAS Number : 127

- 68

- 4

Analysed Concentration : 96.9% ( Refer Certificate of Analysis in Appendix N° 7 )

Batch N° : 50623

Supplied by : Reservol LP

Manufactured by : Tiarco Chemical

Date of Manufacture : August 09, 2011

Date of Expiry : August 09, 2013

Appearance : Yellow to tan granular solid

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

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

supplied by the Sponsor at a temperature between 40 and 90° F in

the Test Item Control Office (TICO)

. The stability of the test item

in storage is the responsibility of the Sponsor.

2.2 Instruments and Equipments

Tattoo Machine : AIMS TM Tattoo Machine

Balance s : Sartorious AG GOTTIENGEN, Germany

(Capable of measuri ng 0.1 g to 2100 g)

Intubation Cannula : Metal Cannula, Size 16 G x 5 cm

Syringe : BD 5 mL Borosilicate Glass Syringe

2.3 Solvent s and Chemical

RO Water : Reverse Osmosis Water F iltration S ystem

Disinfectant Solution : Dettol 2.5 %JRF Study Number: 401-1- 04 -5131 (Final Report) Page 12 of 43

2 .4 Animals

Te st Species : Rat ( Rattus norvegicus )

Strain : Wistar

Animal Source : Animal Breeding Facility, JRF

Total Number of Animals Used : Nine females

Female rats were nulliparous and non-pregnant

Body Weight (g) before Dosing : Minimum: 196

, Maximum: 2 33

Age of the Animals : 8 - 10 weeks at the time of dosing

2.5 Acclimatisation

The rats were received into the experimental room and allowed to acclimatize for a period of 6 to 22

days prior to commencement of dosing.

2.6 Husbandry Practices

Caging : Polypropylene rat cages covered with stainless steel grid top were

used. Autoclaved clean rice husk was used as the bedding material.

Water Bottle : Each cage was supplied with a polypropylene water bottle with a

stainless steel nozzle.

Housing : 1 rat per cage

Room Sanitation : Each day, the floor of the experiment al room was swept and the

floor was mopped and all work tops were wiped with a disinfectant

solution.

2.7 Animal Identification

Each rat was uniquely numbered on the tail using a tattoo machine. Appropriate labels were attached

to the cages indicating the study number, test item code, sex, dose, type of study, cage number and

animal number.

2.8 Feed and Water

Rats were provided with Teklad Certified Global High Fiber Rat/Mice Feed manufactured by Harlan,

USA. The quality of feed and water is regularly monitored at Jai Research Foundation. There were no

known contaminants in the feed and water at levels that would have interfered with the experimental

results obtained.

Feed : Rat pellet feed was provided ad libitum

.

Water : Drinking water filtered through Reverse Osmosis water filtration system

provided ad libitumJRF Study Number: 401-1- 04 -5131 (Final Report) Page 13 of 43

2.9 Environmental Conditions

Animal Room : BMR-9, Department of Toxicology

Temperature Range : 20 to 23 °C

Relative Humidity Range : 65 to 66 %

Photoperiod : The photoperiod was 12 h artificial light and 12 h darkness, light

hours being 06:00 h

–

18:00 h

Air Changes : Minimum 15 air changes/h

2.10 Experimental Activities

The following observations/activities were performed during the experimental period:

Daily

- animal observations

- providing feed and water

- floor sweeping and mopping

- rack cleaning

- recording room temperature and relative humidity

- recording photoperiod

Weekly

- recording body weight

2.11 Dose Preparation

The test item was forming clear solution in distilled water, so the actual dosing solution was prepared

using distilled water as vehicle. Required quantity ( 175 mg for rat N° 1, 310 mg for rat N° 2, 550 mg

for rat N° 3, 990 mg for rat N° 4, 1750 mg for rat N° 5, 3100 mg for rat N°6 and 500 0 mg each for rat

N° 7, 8 and 9) of sodium 3-nitrobenzensulfonate was mixed with distilled water. The total volume of

the dose solution was made upto 10 mL with distilled water

. Dosing solutions were prepared freshly

prior to dosing on all the occasions.

2.12 Preparation of Dosing Solution

A dose volume of 10 mL/kg body weight was maintained for each rat. All rats were dosed by gavage

using a metal cannula attached syringe. Rats were fasted overnight prior to dosing till three hours postdosing.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 14 of 43

2.13 Main Study

This study was conducted as a main study. Rat N° 1 was given a single dose of 175 mg sodium 3-

nitrobenzensulfonate/kg body weight (slope 4). As no mortality was observed at this dose, rat N° 2

was treated with higher dose of 310 mg sodium 3-nitrobenzensulfonate/kg body weight. No mortality

was observed at this dose level, therefore rat N° 3 was treated with higher dose of 550 mg sodium 3-

nitrobenzensulfonate/kg body weight. At this dose level no mortality was observed, hence rat N° 4 was

treated with higher dose of 990 mg sodium 3-nitrobenzensulfonate/kg body weight. No mortality was

observed at this dose level, therefore rat N° 5 was treated with higher dose of 1750 mg sodium 3-

nitrobenzensulfonate/kg body weight. At this dose level no mortality was observed, hence rat N° 6 was

treated with higher dose of 3100 mg sodium 3-nitrobenzensulfonate/kg body weight. As no mortality

was observed at this dose level, rat N° 7 was treated with higher dose of 5000 mg sodium 3-

nitrobenzensulfonate/kg body weight. No mortality was observed at this dose level, therefore rat N° 8

and 9 were treated with 5000 mg sodium 3-nitrobenzensulfonate/kg body weight with gap of 48 h

interval. As no mortality was observed at this dose level, the end point was achieved, and further

testing was not required.

2.14 Observations

The rats were observed for signs of toxicity and mortality at 30 minutes, 1, 2, 3, 4 and 5 h post dosing on

the day of dosing (Appendix 1). Subsequently, the rats were observed twice a day for morbidity and

mortality for a period of 14 days following oral dosing. The clinical signs were recorded once a day

(Table 1and Appendix 1). Individual body weight w as recorded prior to dosing on day 0 and on days 7

and 14 following oral dosing (Appendix 2).

2.15 Pathology

At the end of the 14-day observation period all the rats were euthanised by carbon dioxide asphyxiation

were subjected to a gross pathological examination consisting of an external examination and opening of

abdominal and thoracic cavities. Gross macroscopic changes, if any, were recorded (Appendix 3).JRF Study Number: 401-1- 04 -5131 (Final Report) Page 15 of 43



### 3. RESULTS

3.1 Mortalities

No mortalities were observed in all the animals treated with sodium 3

- nitrobenzensulfonate

(Appendix 1).

3.2 Clinical Observations

No sign of toxicity was observed in all the animals treated with sodium 3

- nitrobenzensulfonate

( Appendix 1)

.

3.3 Body Weight

N ormal gain in body weight was observed in all the animals treated with sodium

3

- nitrobenzensulfonate (Appendix 2).

3.4 Necropsy (Macroscopic Findings)

External

External examination of the terminally sacrificed animals did not reveal any abnormality of

pathological significance ( Appendix 3).

Internal

Visceral examination of terminally sacrificed animals did not reveal any lesion of pathological

significance. In absence of any pathological lesion in terminally sacrificed animals, it is concluded that

the test item did not produce any treatment related effect at the dose level tested in present study

(Appendix 3).

3.5 Interpretation of Results

As per the Dixon‟s maximum likelihood method using software (AOT 425 StatPgm) the calculated

LD 50 is found to be greater than 5000 mg/kg body weight in female Wistar rats.

According to the EPA toxicity categories, sodium 3- ni trobenzensulfonate is classified under

“ Category IV ” and as per Globally Harmonized System (GHS 2011), sodium 3-nitrobenzensulfonate

is classified under “ Category 5 ” (Appendix 5).JRF Study Number: 401-1- 04 -5131 (Final Report) Page 16 of 43



### 4. CONCLUSION

The calculated acute oral median lethal dose of Sodium 3-nitrobenzensulfonate in Wistar Rat as per

the Dixon‟s maximum likelihood method is found to be greater than 5000 mg/kg body weight in

female Wistar rats.

Sodium 3-nitrobenzensulfonate is being classified (Appendix 5) as follows:

Globally Harmonized System (GHS 20 11 ) : Category 5

EPA Toxicity Categories (December 2002) : Category IVJRF Study Number: 401-1- 04 -5131 (Final Report) Page 17 of 43



### 5. REFERENCES

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).

OECD, 2008: OECD N° 425, “Acute Oral Toxicity– Up -and- Down Procedure”, The Organisation for

Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, adopted

by the Council on October 03, 2008.

United Nations, 20 11 : GHS ST/SG/AC.10/30/ Rev. 4, Globally Harmonized System of Classification

and Labelling of Chemicals (GHS)

– Fourth Revised Edition. United Nations, New York and Geneva

ISBN

– 978

- 92 -1- 117042 -9.

US EPA, 2002: OPPTS 870.1000 “Acute Toxicity Te sting

– Background”. United States

Environmental Protection Agency, Health Effects Test Guidelines, EPA 712-C- 02 -189, December

2002.

US EPA, 2002: OPPTS 870.1100 “Acute Oral Toxicity”. United States Environmental Protection

Agency, Health Effects Test Guidelines, EPA 712-C- 02 -190, December 2002.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 18 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

TABLE 1

Test Sequence and Mortalities

Sex: Female

Rat N° Dose

(mg/kg body

weight) Mortality after Dosing at

1⁄2

- 5 h 24 h 48 h 72 h 4

– 7

Day 8

– 14

Day

1 175 O O O O O O

2 310 O O O O O O

3 550 O O O O O O

4 990 O O O O O O

5 1750 O O O O O O

6 3100 O O O O O O

7 5000 O O O O O O

8 5000 O O O O O O

9 5000 O O O O O O

Key: O = SurvivedJRF Study Number: 401-1- 04 -5131 (Final Report) Page 19 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 1

Individual Clinical Observations and Mortalities

Sex: Female

Rat

N° Dose
(mg/kg

body
weight) Clinical Signs Observed after Dosing

A t Hour
(Day

- 0) On Day

1⁄2 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 175 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2 310 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

3 550 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

4 990 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

5 1750 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

6 3100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

7 5000 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

8 5000 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

9 5000 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Key: 1 = Normal

, Day-0 = Day of dosingJRF Study Number: 401-1- 04 -5131 (Final Report) Page 20 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 2

Body Weight (g) and Percent Body Weight Change of Individual Rat

Sex: Female

Rat N° Dose

(mg/kg body

weight) Body Weight (g) on Day Percent Body Weight
Change on Day

0 7 14 0

- 7 0

- 14

1 175 196 230 245 17.35 25. 00

2 310 206 233 236 13.11 14.56

3 550 200 223 230 11.50 15.00

4 990 218 233 240 6.88 10.09

5 1750 201 217 225 7.96 11.94

6 3100 225 232 238 3.11 5.78

7 5000 230 249 264 8.26 14.78

8 5000 199 208 223 4.52 12.06

9 5000 233 250 258 7.30 10.73

Key : 0 = Before dosing

, - = Not applicableJRF Study Number: 401-1- 04 -5131 (Final Report) Page 21 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 3

Necropsy Findings of Individual Rat

Sex: Female

Rat N° Dose

(mg/kg body
weight) Mode of

Death External Internal

1 175 Termin al

s acrifice No abnormality d etected No abnormality detected

2 310 Terminal

s acrifice No abnormality detected No abnormality detected

3 550 Terminal

s acrifice No abnormality detected No abnormality detected

4 990 Terminal

s acrifice No abnormality de tected No abnormality detected

5 1750 Terminal

s acrifice No abnormality detected No abnormality detected

6 3100 Terminal

s acrifice No abnormality detected No abnormality detected

7 5000 Terminal

s acrifice No abnormality detected No abnormality detect ed

8 5000 Terminal

s acrifice No abnormality detected No abnormality detected

9 5000 Terminal

s acrifice No abnormality detected No abnormality detectedJRF Study Number: 401-1- 04 -5131 (Final Report) Page 22 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 4

Dose Progression

S lope = 1 2 3 4 5 6 7 8

0.175* 0.175* 0.175* 0.175* 0.175* 0.175* 0.175* 0.175*

0.24 0.23

0.275 0.26

0.31 0.34 0.31

0.375 0.375

0.41

0.44 0.47

0.55 0.55 0.55 0.55

0.69 0.65

0.73

0. 81 0.82

0.99 0.91 0.97

1.09 1.2

1.26 1.29

1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75

2.4 2.3

2.75 2.6

3.1 3.4 3.1

3.75 3.75

4.4 4.1

4.7

5.5 5.5 5.5 5.5

6.9 6.5

7.3

8.1 8.2

9.9 9.1 9.7

10.9 12

12.6 12.9

17.5 17.5 17.5 17.5 17.5 17.5 17.5 17.5

24 23

27.5 26

31 34 31

* = If lower doses are needed, continue progressions to a lower doseJRF Study Number: 401-1- 04 -5131 (Final Report) Page 23 of 43

APPENDIX 4 (Continued)

Slope = 1 2 3 4 5 6 7 8

37.5 37.5

44 41

47

55 55 55 55

65

69 73

81 82

99 91 97

109 120

126 129

175 175 175 175 175 175 175 175

240 230

275 260

310 340 310

375 375

440 410

470

550 550 550 550

650

690 730

810 820

990 910 970

1090 1200

1260 1290

1750 1750 1750 1750 1750 1750 1750 1750

2400 2300

2750 2600

31 00 3100

3750 3400

4100

5000 5000 5000 5000 5000 5000 5000 5000

Note: Study conducted as a main study with starting dose of 175 mg/kg body weight (Slope 4)JRF Study Number: 401-1- 04 -5131 (Final Report) Page 24 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 5

Classifications

A. According to Globally Harmonized System f or Classification and Labelling of Chemicals

(GHS 20 11 )JRF Study Number: 401-1- 04 -5131 (Final Report) Page 25 of 43

APPENDIX 5 (Continued)JRF Study Number: 401-1- 04 -5131 (Final Report) Page 26 of 43

APPENDIX 5 (Continued)

B. EPA Toxicity Categories

(Source: Health Effects Test Guidelines, OPPTS 870.1000)

Study Category I Category II Category III Category IV

Acute Oral Up to and including

50 mg/kg > 50 through

500 mg/kg > 500 through

5000 mg/kg > 5000 mg/kg

Acute Dermal Up to and including

200 mg/kg > 200 through

2000 mg/kg > 2000 through

5000 mg/kg > 5000 mg/kg

Acute

Inhalation Up to and including

0.05 mg/liter > 0.05 through

0.5 mg/liter > 0.5 through

2 mg/liter > 2 mg/liter

Eye Irritation Corrosive (Irreversible

destruction of ocular

tissue) or corneal

involvement or

irritation persisting for

mor e than 21 days Corneal involvement or

irritation clearing in 8

- 21 days Corneal involvement or

irritation clearing in 7

days or less Minimal effects

clearing in less

than 24 hours

Skin Irritation Corrosive (tissue

destruction into the

dermis and/or scarrin g) Severe irritation at 72

hours (severe erythem a

or edema) Moderate irritation at

72 hours (moderate

erythema) Mild or slight

irritation (no

irritation or

slight erythema)JRF Study Number: 401-1- 04 -5131 (Final Report) Page 27 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 6

Signed Study Plan

JRF Study Number: 401-1- 04

- 5131 Page 1 of 14

STUDY PLAN

ACUTE ORAL TOXICITY STUDY OF

SODIUM 3-NITROBENZENSULFONATE IN RATS

GUIDELINES: U.S. EPA OPPTS 870.1100 and OECD N° 425

SPONSOR

TIARCO CHEMICAL

1300 TIARCO DRIVE

DALTON, GA 30720

TECHNICAL CONTACT : JEFF VANDERLIP

REGULATORY CONTACT : BENNETT KING

STUDY DIRECTOR: DEEPAK UJAWANE

TEST FACILITY

JAI RESEARCH FOUNDATION

DEPARTMENT OF TOXICOLOGY

VALVADA

- 396 108

DIST. VALSAD

GUJARAT

INDIA

AUGUST - 2012JRF Study Number: 401-1- 04 -5131 (Final Report) Page 28 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 2 of 14

CONTENTS

PAGE N°

TITLE PAGE

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

CONTENTS

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

1. GENERAL INFORMATION

................................................................................................................ 3
1.1 Study Professionals

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

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

1.3 Technical Contact

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

...................................................................................................................... 3
1.5 Study Schedule

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

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

2. INTRODUCTION

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

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

................................................................................................................. 4
2.3 Principle of the Test Method

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

2.4 Test Item

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

3. GOOD LABORATORY PRACTICE (GLP)

........................................................................................ 6
3.1 GLP Compliance

......................................................................................................................... 6
3.2 Standard Operating Procedures (SOPs)

....................................................................................... 6
3.3 Amendment to Study Plan

........................................................................................................... 6
3.4 Deviation from the Study Plan

..................................................................................................... 6
3.5 Quality Assurance

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

4. ANIMAL WELFARE

............................................................................................................................ 6
4.1 Humane Endpoint

........................................................................................................................ 7

5. EXPERIMENTAL PROCEDURE

........................................................................................................ 7

5.1 Initial Considerations

............................................................................................................................. 7

5.2 Animals

.................................................................................................................................................. 7

5.2.1 Justification

......................................................................................................................... 7
5.2.2 Specification

....................................................................................................................... 7

5.3 Acclimatisation

............................................................................................................................ 7
5.4 Husbandry Practices

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

5.5 Animal Room Sanitation

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

5.6 Feed and Water

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

5.7 Environmental Conditions

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

5.8 Dose Administration

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

5.9 Number of Animals and Dose Levels

.......................................................................................... 8
5.10 Main Study

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

5.11 Duration

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

5.12 Observations

................................................................................................................................ 9
5.13 Dose Progressions

....................................................................................................................... 10
5.14 Pathology

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

5.15 Evaluation of Result

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

6. REPORT

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

7. ARCHIVES

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

8. REFERENCES

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

9. STUDY PLAN APPROVAL

................................................................................................................. 14JRF Study Number: 401-1- 04 -5131 (Final Report) Page 29 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 3 of 14

1. GENERAL INFORMATION

1.1 Study Professionals

Name of

Personnel Qualificat ion Profession Experience Training

Deepak Ujawane
(Study Director) M. Pharm. Toxicologist 3 years Declared Study Director for
acute toxicity studies

Manish V. Patel Ph.D. Toxicologist 15 years Declared Study Director for
all Development and
reproductive toxicity studies

Mukesh Posia M.V.Sc. Veterinarian 2 years Trained as study veterinarian
for animals handling

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

pathologist

Note: List of additional study personnel will be documented in the study file and will be presented in the final report

1.2 Test Facility Management : Abhay Deshpande, Ph.D.

Global Director,

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

Near Daman Ganga Bridge,

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

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

1.3 Technical Contact : Jeff Vanderlip

706/277- 1300

Tiarco Chemical

1300 Tiarco Drive

Dalton, Georgia 30720

1.4 Regulatory Contact : Bennett King

864/457- 2321

Tiarco Chemical

1300 Tiarco Drive

Dalton, Georgia 30720JRF Study Number: 401-1- 04 -5131 (Final Report) Page 30 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 4 of 14

1.5 Study Schedule

Study Initiation Date : August 10, 2012

Experiment Start Date : August 21, 2012

Experiment Completion : Latest by September 2012

Draft Report Submission : Latest by October 2012

Study Completion : Within two weeks from the date of receipt of comments on the

final draft report from the Sponsor.

1.6 Study Plan and Amendment (if any) Distribution

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

2. INTRODUCTION

2.1 Objective

This study is not intended to calculate a precise LD 50 but to determine the range of exposures where lethality is

expected since death of a proportion of the rats is still the major endpoint of this test. It also provides

information both for hazard assessment and for hazard classification purposes of sodium 3-

nitrobenzensulfonate.

2.2 Regulatory Guidelines

The United States Environmental Protection Agency (December 2002). Health Effects Test Guidelines, OPPTS

870.1100, EPA 712-C- 02

- 190, “Acute Oral Toxicity”;

and

The Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals,

N° 425 “Acute Oral Toxicity – Up -and- Down Procedure”, adopted by the Council on Octobe r 03, 2008.

2.3 Principle of the Test Method

Animals will be dosed, one at a time, at a minimum of 48-hour intervals. The first animal will receive the dose a

step below the level of the best estimate of the LD 50 . If the animal survives, the dose for the next animal will be

increased by a factor (e.g. one half log times the original dose). If it dies, the dose for the next animal will be

decreased by a similar dose progression (refer 5.13 for a dose progression, e.g. one half log unit). The decision

whether and how much to dose the next animal will be based on the 48-hour survival pattern of all the rats up to

that time. The testing stops when one of the following stopping criteria will meet first:JRF Study Number: 401-1- 04 -5131 (Final Report) Page 31 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 5 of 14

(a) 3 consecutive animals survive at the upper bound;

(b) 5 reversals occur in any 6 consecutive animals tested;

(c) At least 4 animals have followed the first reversal and the specified likelihood ratios exceed

the critical value.

Dosing will be stop when one of these criteria is satisfied, at which time an estimate of the LD 50 and a

confidence interval are calculated for the test based on the status of all the animals at termination. For most

applications, testing will be completed with only 4 animals after initial reversal in animal outcome.

The LD 50 is calculated using the Dixon‟s maximum likelihood method using (AOT 425 StatPgm) software. The

results of the main test procedure serve as the starting point for a computational procedure to provide a

confidence interval estimate where feasible.

2.4 Test Item

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

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

otherwise instructed by the Sponsor. The details relating to the test item (as furnished by the study Sponsor) are

as follows:

Test Item Name : Sodium 3

- nitrobenzensulfonate

IUPAC Name : Benzenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CA Name : Ben zenesulfonic acid, 3

- nitro

- , sodium salt (1:1)

CAS Number : 127

- 68

- 4

Analysed Concentration : 96.9% (Information provided by the Sponsor via CoA)

Batch N° : 50623

Supplied by : Reservol LP

Manufactured by : Tiarco Chemical

Date of Manufacture : August 09 , 2011

Date of Expiry : August 09, 2013

Appearance : Yellow to tan granular solid

Data related to the characterization of the test item are documented in a Certificate of Analysis provided by the

Sponsor and will be placed in annexes of the study report. The test item information and analytical identification

provided by the Sponsor will be responsibility of the Sponsor.

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

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

the Sponsor at a temperature between 40 and 90 °F in the Test Item

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

responsibility of the Sponsor.

JRF Test Item Code : SNB 444JRF Study Number: 401-1- 04 -5131 (Final Report) Page 32 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 6 of 14

3. GOOD LABORATORY PRACTICE (GLP)

3.1 GLP Compliance

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

in 1997), ENV/MC/CHEM (98)17, N° 1, Environment Directorate, the Organisation for Economic Cooperation and Development, Paris (1998) and all subsequent OECD consensus documents.

3.2 Standard Operating Procedures (SOPs)

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

Procedures of Jai Research Foundation.

3.3 Amendment to Study Plan

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

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

approval.

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

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

soon as possible by way of formal study plan amendment.

3.4 Deviation from the Study Plan

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

3.5 Quality Assurance

This study plan has been audited by JRF QAU and documented (Number 47265). The Quality Assurance Unit

(QAU) of JRF will audit/inspect the critical phase(s) of the study, the raw data, draft and final reports. The

audit/inspection reports will be provided to the Study Director and the Test Facility Management. The date of

audits/inspections and reporting of findings to the Study Director and the Test Facility Management will be

incorporated in the study report.

4. ANIMAL WELFARE

The study will be undertaken in compliance with the „Guidelines for Laboratory Animals Facility‟ issued by the

Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. These

guidelines promote the humane care of animals used in research by providing specifications that will enhance

animal well being and experimental quality for the advancement of biological knowledge that is relevant to

humans and animals.

Jai Research Foundation is committed to 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.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 33 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 7 of 14

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

(IAEC), Jai Research Foundation.

JRF is also accredited with Association for Assessment and Accreditation of Laboratory Animal Care

(AAALAC) that promotes the humane treatment of animals in science.

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. The end point of this study is death or impending death.

5. EXPERIMENTAL PROCEDURE

5.1 Initial Considerations

The test item, at doses that cause marked pain and distress due to corrosive or severely irritant actions, will not

be administered and, if required, the study will be terminated. Animals obviously in pain or showing signs of

severe and enduring distress will be humanely killed and considered in the interpretation of the test results.

5.2 Animals

5.2.1 Justification

Rat is selected as a test system because it is a readily available laboratory rodent species. It has been historically

shown to be a suitable model for acute oral toxicity assessment and is recommended by the EPA, OECD and

other regulatory authorities. The results of the study may be of value in predicting the toxicity of the test item to

human being.

5.2.2 Specification

Healthy, young adult Wistar rats ( Rattus norvegicus ) will be obtained from the Animal Breeding Facility, Jai

Research Foundation and will be used for the study. Female rats will be nulliparous and non-pregnant. At the

initiation of dosing, the rats will be between 8 and 12 weeks. The weight range of the rats will be within ± 20%

of the mean body weight of any previously dosed rats at the time of dosing.

5.3 Acclimatisation

The rats after veterinary examination for good health will be received into the experimental room and allowed to

acclimatise to the laboratory conditions for a minimum period of 5 days prior to commencement of dosing.

Finally, the rats will be checked once again before commencing the experiment.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 34 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 8 of 14

5.4 Husbandry Practices

The rats will be housed individually in polypropylene rat cages layered with clean sterilized rice (paddy) husk as

the bedding. Cage will be supplied with a polypropylene water bottle fitted with a sterilized steel nozzle.

Individual rats will be identified with unique numbers tattooed on tail using a tattoo machine. The cages will be

labelled with details of the study number, test item code, sex, dose, type of study, cage number and animal

numbers.

5.5 Animal Room Sanitation

Each day, the floor of the experimental room will be swept and all worktops and the floor will be mopped with a

disinfectant solution.

5.6 Feed 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. However, rats

will be fasted over-night prior to dosing and 3 to 4 h after dosing. Fresh feed will be provided at least once a

week.

5.7 Environmental Conditions

The temperature of the experimental room will be maintained at 22 ± 3 °C and the relative humidity between 30

and 70 percent. The photoperiod will be 12 h light and 12 h darkness, light hours being 06:00 - 18:00 h

approximately and air changes will be minimum 15/hour.

5.8 Dose Administration

Single oral dosing will be done by using intubation cannula. Test item will be mixed with a vehicle. Fresh dose

solutions will be prepared and administered to test system within 2 hours of preparation. The concentration of

the test item will vary so as to permit constant volume dosing. The total volume administered will not exceed 10

mL/kg body weight.

5.9 Number of Animals and Dose Levels

Single rat will be dosed in sequence at an interval of not less than 48 h. However, the time intervals between

dosing are determined by the onset, duration, and severity of toxic signs. The first rat will be dosed at 175

mg/kg body weight. If the rat survives, the second rat will receive a higher dose. If the first rat dies or appears

moribund, the second rat will receive a lower dose. The starting dose level will be 175 mg/kg body weight

(Table 1

–

Slope 4).

5.10 Main Study

First rat will be dosed at 175 mg/kg body weight of sodium 3-nitrobenzensulfonate. If first animal survives,

second animal will receive a higher dose. If first animal dies, second animal will receive a lower dose and the

dose will be selected from the Slope 4. Using the slope N° 4, doses may be selected from the sequence 55, 99,

175, 310, 550, 990, 1750, 3100 and 5000.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 35 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 9 of 14

5.11 Duration

The rats will be observed for a minimum period of 14 days following dose administration. The observation

period will be extended if delayed toxic effects are apparent.

5.12 Observations

After oral intubation on the day of dosing (day 0), death and signs of toxicity will be recorded at least at 30

minutes, 1, 2, 3 and 4 hours and once thereafter dosing. Subsequently, the rats will be observed twice a day for

mortality and moribund state, if any. Clinical signs will be recorded once a day. Clinical signs after treatment

will be recorded in terms of time of onset, duration, intensity and subsequent course of the reactions.

Observations will include, but not be limited to evaluation of skin and fur, eyes and mucous membranes,

respiratory and circulatory effects, autonomic effects and central nervous system effects, behavioural pattern,

somatomotor activity and observation of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body weight will be recorded individually, immediately prior to dosing (day 0) and on days 7 and 14 after

dosing and at death (for found dead animals). Weekly body weight changes will be calculated and reported.

Depending on the number of humanely killed or found dead rats, the test procedure to be followed is as

indicated.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 36 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 10 of 14

5.13 Dose Progressions

Table 1: Choose a Slope and Read Down the Column (all doses in mg/kg body weight)

Slope = 1 2 3 4 5 6 7 8

0.175* 0.175* 0.175* 0.175* 0.175* 0.175* 0.175* 0.175*

0.24 0.23

0.275 0.26

0.31 0.34 0.31

0.375 0.375

0.41

0.44 0.47

0.55 0.55 0.55 0.55

0.69 0.65

0.73

0.81 0.82

0.99 0.91 0.97

1.09 1.2

1.26 1.29

1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75

2.4 2.3

2.75 2.6

3 .1 3.4 3.1

3.75 3.75

4.4 4.1

4.7

5.5 5.5 5.5 5.5

6.9 6.5

7.3

8.1 8.2

9.9 9.1 9.7

10.9 12

12.6 12.9

17.5 17.5 17.5 17.5 17.5 17.5 17.5 17.5

24 23

27.5 26

31 34 31

* = If lower doses are needed, continue progressions to a lower doseJRF Study Number: 401-1- 04 -5131 (Final Report) Page 37 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 11 of 14

Dose Progressions (Continued)

Slope = 1 2 3 4 5 6 7 8

37.5 37.5

44 41

47

55 55 55 55

65

69 73

81 82

99 91 97

109 120

126 129

175 175 175 175 175 175 175 175

240 230

275 260

310 340 310

375 375

440 410

470

550 550 550 550

650

690 730

810 820

990 910 970

1090 1200

1260 1290

1750 1750 1750 1750 1750 1750 1750 1750

2400 2300

2750 2600

3100 3100

3750 3400

4100

5000 5000 5000 5000 5000 5000 5000 5000JRF Study Number: 401-1- 04 -5131 (Final Report) Page 38 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 12 of 14

5.14 Pathology

The rats which die during the course of the study will be subjected to gross pathological examination at the

earliest, not exceeding 24 h since found dead. The surviving rats sacrificed (CO 2 asphyxiation) terminally will

be subjected to gross pathological examination immediately. Gross macroscopic changes will be recorded for all

the rats.

5.15 Evaluation of Result

All raw data will be recorded in standard formats. The software program AOT 425 StatPgm (JRF/SSI/SOP/957)

will be used for performing the test and calculating the LD 50 and confidence level. The results of the main test

procedure serve as the starting point for a computational procedure to provide a confidence interval estimate

where feasible. The results of the limit test will be evaluated as per the guidelines. The test item will be

classified based on EPA Toxicity Categories (December 2002) and Globally Harmonised System (GHS 2011).

6. REPORT

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

in PDF. The report will include the following information:

- the test item details e.g. source, batch N°, purity etc.

- vehicle details (if any)

- species/strain/source, number, age and sex of the test animal, feed and environmental conditions

- description of caging condition e.g. N° of rat per cage, bedding material, photoperiod and

identification of feed of test rat

- full details of the experimental procedures

- a summary of observations, including tabulation of signs of toxicity to treatment and dose level and

time of individual death (if any)

- time course of onset of signs of toxicity and whether these were reversible for each animal

- gross pathological findings,

- body weight data (individual weight of rat on the day of dosing, in weekly intervals, at the time of

death or sacrifice and body weight changes)

- discussion and interpretation of results (LD 50 data, statistical treatment of results)

- classification according to EPA Toxicity categories (December 2002) and classification according to

Globally Harmonised System (GHS 2011).

- conclusion

- personnel involved in the study

- signed study plan and study plan amendment(s) (if any)

- record of deviation(s) from the study plan (if any)JRF Study Number: 401-1- 04 -5131 (Final Report) Page 39 of 43

APPENDIX 6 (Continued)

JRF Study Number: 401-1- 04

- 5131 Page 13 of 14

7. 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. 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.

8. REFERENCES

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).

OECD, 2008: OECD N° 425, “Acute Oral Toxicity– Up -and- Down Procedure”, The Organisation for

Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals, adopted by the

Council on October 03, 2008.

United Nations, 2011: GHS ST/SG/AC.10/30/ Rev.4, Globally Harmonized System of Classification and

Labelling of Chemicals (GHS)

– Fourth Revised Edition. United Nations, New York and Geneva ISBN

– 978

-

92 -1- 117042

- 9.

US EPA, 2002: OPPTS 870.1000 “Acu te Toxicity Testing

– Background”. The United States Environmental

Protection Agency, Health Effects Test Guidelines, EPA 712-C- 02 -189, December 2002.

US EPA, 2002: OPPTS 870.1100 “Acute Oral Toxicity”. The United States Environmental Protection Agency,

Health Effects Test Guidelines, EPA 712-C- 02 -190, December 2002.JRF Study Number: 401-1- 04 -5131 (Final Report) Page 40 of 43

APPENDIX 6 (Continued)JRF Study Number: 401-1- 04 -5131 (Final Report) Page 41 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 7

Certificate of Analysis of Sodium 3-nitrobenzensulfonateJRF Study Number: 401-1- 04 -5131 (Final Report) Page 42 of 43



### Acute Oral Toxicity Study of Sodium 3-nitrobenzensulfonate in Rats

APPENDIX 8

GLP Endorsement of ComplianceJRF Study Number: 401-1- 04 -5131 (Final Report) Page 43 of 43

APPENDIX 8 (Continued)