REPORT 

LEU CO SULFUR BLACK 1 

Acute Oral Toxicity Study in Rats 

Study Director: 

Test Facility: 

Sponsor: 

G. Arcelin 

Harlan Laboratories Ltd. 

Wolferstrasse 4 

4414 Fiillinsdorf I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 11 Switzerland 

Study Identification: Harlan Laboratories Study C31043 

Version: Final 

Study Completion Date: 24-Aug-2009 

Page 1 of 46 

CONTAINS NO CBI 

harlanTM .... 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

Report 

CONTENTS 

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

STATEMENT OF COMPLIANCE ...................................................................................... 4 

QUALITY ASSURANCE GLP STATEMENT ................................................................... 5 

PREFACE ............................................................................................................................. 6 

General Information .. .. . . . . . . . .. . . . .. .. ... . . . .. .. . .. .. . . . .. . . . . . . . .. . . . . . . . ... . . . . . . . . . . . .. ... . . .. . . . .. . . . .. . . . .. . . . .. . . . . 6 

Responsibilities .. . .. .. . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . .. . . . .. . . ... . . . .. . . . .. . .. . .. .. . .. . . . .. . . . . . . . . .. . . . ... .. ... .. ... .. . 6 

Schedule ......................................................................................................................... 6 

Data Requirements I Test Guidelines ............................................................................. 7 

Animal Welfare .............................................................................................................. 7 

Archiving . . . . .. . . .. . . . .. . . . . . . . .. . .. . . .. . . . . . . . .. . .. . . . .. .. .. . .. .. . .. . . . .. .. . .. . . . . . . .. . .. .. .. . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . 7 

1 SUMMARY ....................................................................................................................... 8 

2 PURPOSE .......................................................................................................................... 9 

3 MATERIALS AND METHODS ..................................................................................... I 0 

3.1 TestSystem ............................................................................................................ 10 

3.2 Allocation ............................................................................................................... I 0 

3.3 Husbandry .............................................................................................................. II 

3.4 Test Item and Vehicle ............................................................................................ II 

3.4.1 Test Item ....................................................................................................... II 

3.4.2 Vehicle ......................................................................................................... 12 

3.5 Preparation ofDose Formulations .......................................................................... I2 

3.6 Test Item Administration ....................................................................................... 13 

3. 7 Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 

3.8 Observations ........................................................................................................... 13 

3.9 Pathology ................................................................................................................ 13 

3.9.1 Necropsy ...................................................................................................... 13 

3.1 0 Statistical Analysis .. .. .... . .... .. .. .. .. .. .. .. .. . .. .. .. .. .. .... .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .... .. .. .. ...... .. .. 13 

3.11 Data Compilation .. .. ............ ...... .. .. .. .. .. ...... .. .. .. .... .. .. .. .. .. .... .. .. .. .. .. .. .. .... .. .... .... .. .... . 14 

4 RESULTS AND DISCUSSION ...................................................................................... 15 

4.1 Observations ........................................................................................................... 15 

4.1.1 Viability I Mortality ..................................................................................... 15 

4.1.2 Clinical Signs ............................................................................................... 15 

4.1.3 Body Weights ............................................................................................... 15 

4.2 Pathology ................................................................................................................ 15 

4.2.1 Macroscopic Findings .............................................................................. .... 15 

5 CONCLUSION ................................................................................................................ 16 

6 INDIVIDUAL TABLES .................................................................................................. 17 

Clinical Signs ............................................................................................................... 18 

Body Weights .. . .. . . .. . . . . . . . .. . . . .. . . . .. .. . .. .. . .. .. . .. . . .. . . ... . . .. . .. .. .. . . . .. . . . . . .. . . . . . . . .. . . . .. .. ... .. . .. . . ... . . . . . . . l 9 

Macroscopic Findings .................................................................................................. 20 

APPENDIX I - STUDY PLAN AND AMENDMENT ...................................................... 22 

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Leuco Sulfur Black 1 

Report 

Study Plan ........................................................................................................... ......... 23 

First Amendment to Study Plan ................................................................................... 38 

09009e53&06cb3ab 3 
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Leuco Sulfur Black 1 

GOOD LABORATORY PRACTICE 

Report 

STATEMENT OF COMPLIANCE 

Harlan Laboratories Study: 

Test Item: 

Study Director: 

Study Title: 

C31043 

Leuco Sulfur Black 1 

G. Arcelin 

Acute Oral Toxicity Study in Rats 

The formulation trials were performed before the study initiation date. Therefore, they are 

excluded from this statement. 

This study has been performed in compliance with the Swiss Ordinance relating to Good 

Laboratory Practice, adopted May 18th, 2005 (SR 813.112.1]. This Ordinance is based on the 

OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 

1997 by decision ofthe OECD Council [C(97)186/Final]. 

There were no circumstances that may have affected the quality or integrity of the data. 

Study Director: 

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Report 

QUALITY ASSURANCE GLP STATEMENT 

Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen I Switzerland 

Harlan Laboratories Study: 

Test Item: 

Study Director: 

Study Title: 

C31043 

Leuco Sulfur Black 1 

G. Arcelin 

Acute Oral Toxicity Study in Rats 

The general facilities and activities are inspected at least once a year and the results are reported 

to the responsible person and the management. 

Study procedures, with exception of the formulation trials, were periodically inspected. The 

study plan and this report were audited by the Quality Assurance. The dates are given below. 

Dates of Reports to 

Dates and Types of QA Inspections the Study Director 

and Test Facility 

Management 

14-Jan-2009 Study Plan 14-Jan-2009 

21-Jan-2009 Process Based (Test System, Test Item, 21-Jan-2009 

Raw Data, Dose Preparation, Treatment) 

03-Apr-2009 Report 03-Apr-2009 

This statement also confirms that this final report reflects the raw data . 

Quality Assurance: .{or S. van Dongen 

... 

. A/.dM ... h/.L .......... . Date: 2.-1 _...,__,.._ 2..0o 5 

09009c53806d5407 5 
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Leuco Sulfur Black 1 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Laboratory I Technical Coordinator: 

Quality Assurance: 

HeadofQA: 

09009e53806d5407 

Report 

Leuco Sulfur Black 1 

Acute Oral Toxicity Study in Rats 

Clariant Produkte (Schweiz) AG 

Rothausstrasse 61 

4132 Muttenz I Switzerland 

Dr. Thomas Roth 

SCCGmbH 

Mikroforum Ring 1 

55234 Wendelsheim I Germany 

Harlan Laboratories Ltd. 

Wolferstrasse 4 

4414 Fiillinsdorf I Switzerland 

Harlan Laboratories Ltd. 

Quality Assurance GLP 

Zelgliweg 1 

4452 Itingen I Switzerland 

Dr. C. Simon until 08-Jul-2009 

G. Arcelin from 09-Jul-2009 

G. Arcelin until 08-Jul-2009 

E. Rached from 09-Jul-2009 

C. Weng 

T. Fink 

6 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

Schedule 

Experimental Starting Date: 

Termination (Necropsy): 

Delivery of Animals: 

Acclimatization: 

Administration I Treatment: 

Experimental Completion Date: 

Report 

20-Jan-2009 

10-Feb-2009 (females, 2319 mg/kg) 

12-Feb-2009 (females, 2319 mglkg) 

20-Jan-2009 (females, 2319 mglkg) 

22-Jan-2009 (females, 2319 mglkg) 

20-Jan-2009 to 26-Jan-2009 (females, 2319 mglkg) 

22-Jan-2009 to 28-Jan-2009 (females, 2319 mglkg) 

27-Jan-2009 (females, 2319 mglkg) 

29-Jan-2009 (females, 2319 mg/kg) 

12-Feb-2009 

Data Requirements I Test Guidelines 

This study followed the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

• OECD Guidelines for the Testing of Chemicals, Number 423 "Acute Oral Toxicity -

Acute Toxic Class Method", adopted 17th December 2001. 

• Commission Regulation (EC) No 44012008 of 30 May 2008 laying down test methods 

pursuant to Regulation (EC) No 190712006 of the European Parliament and of the 

Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals 

(REACH), B. I tris. ACUTE ORAL TOXICITY - ACUTE TOXIC CLASS METHOD 

(Official Journal No L 142,3110512008 p. 0158-0173). 

Animal Welfare 

This study was performed in an AAALAC-accredited laboratory in accordance with the Swiss 

Animal Protection Law under license no. 34. 

Archiving 

Harlan Laboratories Ltd. ( 4452 Itingen I Switzerland) will retain the study plan, raw data, sample 

of test item(s) and the final report of the present study for at least ten years. No data were 

discarded without the Sponsor's written consent. 

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1 SUMMARY 

Report 

Two groups, each of three female HanRcc:WIST (SPF) rats, were treated with Leuco Sulfur 

Black 1 by oral gavage administration at a dosage of 2319 mg active ingredient I kg body weight 

(corresponding to 4396 mg test item I kg body weight). The test item was formulated in purified 

water according to the content of the active ingredient at a concentration of 0.439 g/mL and 

administered at a dosing volume of 10 mL/kg. 

The animals were examined daily during the acclimatization period and mortality, viability and 

clinical signs were recorded. All animals were examined for clinical signs during the first 30 

minutes and approximately 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test 

days 2-15. Mortality /viability was recorded during the first 30 minutes, and approximately 1, 2, 3 

and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during 

days 2-15. Body 

weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were 

necropsied and examined macroscopically. 

No deaths occurred during the study. 

Except for black stained feces from test day 2 up to test day 5 at the latest, no clinical signs were 

noted in any animals during the observation time. 

The body weight of the animals was within the range commonly recorded for this strain and age. 

No macroscopic findings were observed at necropsy. 

The median lethal dose of Leuco Sulfur Black 1 after single oral administration to female rats, 

observed over a period of 14 days is: 

LD50 (female rat): greater than 2319 mg active ingredient I kg body weight 

(corresponding to 4396 mg test item I kg body weight) 

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Leuco Sulfur Black 1 

2 PURPOSE 

Report 

The purpose of this study was to assess the acute toxtctty of Leuco Sulfur Black 1 when 

administered by a single oral gavage to rats, followed by an observation period of 14 days. 

This study provides information for both hazard assessment and hazard classification purposes. 

09009e53806d5407 9 
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Report 

3 MATERIALS AND METHODS 

3.1 Test System 

Animals: 

Rationale: 

Breeder: 

Number of Animals per Group: 

Total Number of Animals: 

Age (when treated): 

Body Weight Range (when treated): 

Identification: 

Randomization: 

Acclimatization: 

3.2 Allocation 

The animals were distributed as follows: 

Rat, HanRcc: WIST(SPF) 

Recognized by international guidelines as a 

recommended test system. 

Harlan Laboratories Ltd. 

Laboratory Animal Services 

Wolferstrasse 4 

4414 Fiillinsdorf I Switzerland 

3 females 

6 females 

11 weeks 

175.0 g- 197.7 g 

Unique cage number and corresponding color-coded 

spots on the tail. The animals were marked at 

acclimatization start. 

Selected by hand at time of delivery. 

No computer generated randomization program. 

Under laboratory conditions, after health 

examination. Only animals without any visible signs 

of illness were used for the study. 

Dose Level Group Animal Number 

2319 mg active ingredient I kg 

( 4396 mg test item I kg) 1-3 

2319 mg active ingredient I kg 

( 4396 mg test item I kg) 2 4-6 

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Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

3.3 Husbandry 

Room Numbers: 

Conditions: 

Accommodation: 

Diet: 

Water: 

3.4 Test Item and Vehicle 

3.4.1 Test Item 

Report 

0105 I Harlan Laboratories Ltd., Fiillinsdorf 

Standard Laboratory Conditions. Air-conditioned 

with 10-15 air changes per hour, and continuously 

monitored environment with ranges for room 

temperature 22 ± 3 oc and for relative humidity 

between 30-70% (values above 70% during cleaning 

process possible), automatically controlled light 

cycle of 12 hours light and 12 hours dark, music 

during the daytime light period. 

In groups of three in Makrolon type-4 cages with 

wire mesh tops and standard softwood bedding 

('Lignocel' Schill AG, 4132 Muttenz I Switzerland). 

Pelleted standard Provimi Kliba 3433 rat/mouse 

maintenance diet, batch no. 61108 (Provimi Kliba 

AG, 4303 Kaiseraugst I Switzerland) ad libitum 

(except for the overnight fasting period prior to 

intubation and approximately 3-4 hours post dose). 

Results of analyses for contaminants are archived at 

Harlan Laboratories Ltd. 

Community tap water from Fiillinsdorf ad libitum. 

Results of bacteriological, chemical and contaminant 

analyses are archived at Harlan Laboratories Ltd. 

The following information was provided by the Sponsor: 

Identification: 

CAS No.: 

Description: 

Batch Number: 

Composotion: 

09009e53806d5407 

Leuco Sulfur Black 1 

66241-11-0 

Thixothrophic, black, wet paste 

ESA0007595 

Leuco Sulfur Black 1 

(calculated by amount of bycompounds): 

2,4-Dinitrochlorobenzene: 

2,4-Dinitrophenole: 

Sulfate: 

Chloride: 

52.75% (wlw) 

<20ppm 

<20 ppm 

970 ppm 

750 ppm 

11 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

Stability ofTest Item: 

Expiry Date: 

Stability of Test Item Dilution: 

Storage Conditions: 

Safety Precautions: 

3.4.2 Vehicle 

Report 

Sodium: 

Water (by Karl-Fischer 

titration): 

Stable under storage conditions. 

11-Jul-2011 

> 4h in water. 

1.3% (w/w) 

45.75% (w/w) 

At room temperature (range of20 ± 5 °C), light 

protected. 

Routine hygienic procedures were used to ensure the 

health and safety of the personnel. 

Purified water was found to be a suitable vehicle. 

Purified water was prepared at Harlan Laboratories Ltd. ( deionised water which was processed 

and treated by the PURELAB Option-R unit. This latter links four purification technologies: 

reverse osmosis, adsorption, ion-exchange and photo oxidation). 

The vehicle was chosen after a non-GLP solubility trial which was performed before the study 

initiation date. This formulation trial is excluded from the statement of compliance. 

3.5 Preparation of Dose Formulations 

The dose levels are in terms of the active ingredient (Leuco Sulfur Black 1; 52.75% w/w) in 

the test item. 

The dose formulations were prepared shortly before each dosing occasion using a magnetic 

stirrer and a spatula (Janke & Kunkel, 79219 Staufen I Germany) as homogenizers. 

The test item was weighed into a tared glass beaker on a suitable precision balance and the 

vehicle added (weight:volume). 

Homogeneity of the test item in the vehicle was maintained during administration using a 

magnetic stirrer. 

09009e53806d5407 12 
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Leuco Sulfur Black I 

3.6 Test Item Administration 

Report 

The animals received a single dose of the test item by oral gavage administration at 2319 mglkg 

body weight active ingredient (corresponding to 4396 mg/kg of the test item delivered; based on 

the amount of 52.75% (w/w) Leuco sulphur black 1) after being fasted for approximately 15 to16 

hours (access to water was permitted). Food was provided again approximately 3 hours after 

dosing. 

The dosing volume was 10 mL/kg body weight. 

3. 7 Rationale 

Oral administration was considered to be an appropriate application method as it is a possible 

route of human exposure during manufacture, handling and use ofthe test item. 

3.8 Observations 

Viability I Mortality: 

Clinical Signs: 

Body Weights: 

3.9 Pathology 

3.9.1 Necropsy 

Daily during the acclimatization period, during the 

first 30 minutes and at approximately 1, 2, 3 and 5 

hours after administration on test day 1 (in common 

with the clinical signs) and twice daily during days 

2-15. 

Daily during the acclimatization period, during the 

first 30 minutes and at approximately 1, 2, 3 and 5 

hours after administration on test day 1, depending 

on the occurrence of clinical signs of toxicity. Once 

daily during days 2-15. 

On test days 1 (prior to administration), 8 and 15. 

All animals were killed at the end of the observation periods by carbon dioxide asphyxiation and 

discarded after macroscopic examinations were performed. No organs or tissues were retained. 

3.10 Statistical Analysis 

No statistical analysis was used. 

09009e53806d5407 13 
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Leuco Sulfur Black I 

3.11 Data Compilation 

Body weights were recorded on-line. 

Report 

Viability I mortality and clinical signs were recorded on data sheets. 

Macroscopic findings were compiled into the RCC Tox Computer System during recording. 

The RCC Tox Computer System (RCC-Tox-Lims) had been validated with respect to data 

collection, storage and retrievability. 

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Report 

4 RESULTS AND DISCUSSION 

4.1 Observations 

4.1.1 Viability I Mortality 

No deaths occurred during the study. 

4.1.2 Clinical Signs 

(See Individual Table on p. 16 ) 

For the first group of animals treated at 2319 mg active ingredient I kg (corresponding to 4396 

mg test item I kg), no clinical signs were observed in any of the animals on test 1. On test day 2, 

all three females were noted with black-stained feces and the cage of the animals was changed to 

facilitate further assessment. All animals were free of any signs from test day 3 until the end of 

the study on test day 15. The second group of animals treated at 2319 mg active ingredient I kg 

(corresponding to 4396 mg test item I kg) were free of clinical signs on test day 1 as well. On test 

day 2 up to test day 5 all animals were observed with black-stained feces. On each of these 4 

days the animals were moved to new cages. All animals were free of any clinical signs from test 

day 6 until the end of the study. 

4.1.3 Body Weights 

(See Individual Tables on p. 19 ) 

The body weight ofthe animals was within the range commonly recorded for this strain and age. 

4.2 Pathology 

4.2.1 Macroscopic Findings 

(See Individual Tables on p. 20 ) 

No macroscopic findings were observed at necropsy. 

09009e53806d5407 15 
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Leuco Sulfur Black l 

5 CONCLUSION 

Report 

The median lethal dose of Leuco Sulfur Black 1 after single oral administration to female rats, 

observed over a period of 14 days is: 

09009e53806d5407 

LD50 (female rat): greater than 2319 mg active ingredient I kg body weight 

(corresponding to 4396 mg test item I kg body weight) 

16 
Harlan Laboratories Study C31043 

Leuco Sulfur Black I 

6 INDIVIDUAL TABLES 

09009e53806d5407 

Report 

17 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black 1 

Clinical Signs 

Dose Animg/kg mal Sex Signs 

bw No. 

2319 1 F No clinical signs 

Black -stained 

feces 

2 F No clinical signs 

Black -stained 

feces 

3 F No clinical signs 

Black -stained 

feces 

2319 4 F No clinical signs 

Black -stained 

feces 

5 F No clinical signs 

Black-stained 

feces 

6 F No clinical signs 

Black-stained 

feces 

Key: .J noted 

Report 

Test days 

1 2 

0.5* I* 2* 3* 5* 

..} ..} ..} ..} ..} 

..} 

..} ..} ..} ..} ..} 

..} 

..} ..} ..} ..} ..} 

..} 

..} ..} ..} ..} ..} 

..} 

..} ..} ..} ..} ..} 

..} 

..} ..} ..} ..} ..} 

..} 

3 4 5 6 7 8 9 10 11 12 13 

..} ..} ..} ..} ..} v ..} ..} ..} ..} ..} 

..} ..} ..} ..} ..} v ..} ..} ..} ..} ..} 

..} ..} ..} ..} ..} ..} ..} ..} ..} ..} ..} 

..} ..} ..} ..} ..} ..} ..} ..} 

..} ..} ..} 

..} ..} ..} ..} ..} ..} ..} ..} 

..} ..} ..} 

..} ..} ..} ..} ..} ..} ..} ..} 

..} ..} ..} 

* Examinations were performed within the first 30 minutes and 1, 2, 3 and 5 hours after treatment. 

No clinical signs were evident in any animal during the acclimatization period. 

14 15 

..} ..} 

..} ..} 

..} ..} 

..} ..} 

..} ..} 

..} ..} 

Note: 2319 mg active ingredient I kg body weight (i.e. 4396 mg test item I kg body weight) were 

applied. 

09009e53806d5407 18 
Harlan Laboratories Study C3l 043 

Leuco Sulfur Black I 

BODY WEIGHTS (GRAM) 

GROUP SEX ANIMAL 

GROUP 1 I FEMALES (2319 mg active 

1 

2 

3 

MEAN 

ST.DEV. 

N 

GROUP 2 I FEMALES (2319 mg active 

4 

5 

6 

MEAN 

ST.DEV. 

N 

09009e53806b664f 

Report 

DAY 1 DAY 8 DAY 15 

ingredient/kg; 4396 mg test item/kg) 

197.7 220.6 232.6 

197.3 223.9 228.5 

191.3 213.0 218.2 

195.4 219.2 226.4 

3.6 5.6 7. 4 

3 3 3 

ingredient/kg; 4396 mg test item/kg) 

18 6. 7 201.5 204.7 

180.2 207.1 214.3 

175.0 199.6 208.4 

180.6 202.8 209.2 

5. 9 3.9 4.8 

3 3 3 

19 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

MACROSCOPICAL FINDINGS 

FEMALES 

Report 

GROUP 1 (2319 mg active ingredient/kg; 4396 mg test item/kg) 

ANIMAL 1 (SCHEDULED NECROPSY, 10-FEB-09, DAY 15 AFTER TREATMENT) 

NO FINDINGS NOTED 

ANIMAL 2 (SCHEDULED NECROPSY, 10-FEB-09, DAY 15 AFTER TREATMENT) 

NO FINDINGS NOTED 

ANIMAL 3 (SCHEDULED NECROPSY, 10-FEB-09, DAY 15 AFTER TREATMENT) 

NO FINDINGS NOTED 

09009e53806b6627 20 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black 1 

MACROSCOPICAL FINDINGS 

FEMALES 

Report 

GROUP 2 (2319 mg active ingredient/kg; 4396 mg test item/kg) 

ANIMAL 4 (SCHEDULED NECROPSY, 12-FEB-09, DAY 15 AFTER TREATMENT) 

NO FINDINGS NOTED 

ANIMAL 5 (SCHEDULED NECROPSY, 12-FEB-09, DAY 15 AFTER TREATMENT) 

NO FINDINGS NOTED 

ANIMAL 6 (SCHEDULED NECROPSY, 12-FEB-09, DAY 15 AFTER TREATMENT) 

NO FINDINGS NOTED 

09009e53806b6627 21 
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Leuco Sulfur Black I 

Report 

APPENDIX I -STUDY PLAN AND AMENDMENT 

09009e53806b66a2 22 
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Leuco Sulfur Black 1 

STUDY PLAN 

Report 

LEUCO SULFUR BLACK 1 

09009e53806cb39a 

Acute Oral Toxicity Study in Rats 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

Version: 

Dr. C. Simon 

Harlan Laboratories Ltd. 

Wolferstrasse 4 

4414 Fullinsdorf I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

CH-4132 Muttenz 1/ Switzerland 

Harlan Laboratories Study C31 043 

Final 

Page 1 of 15 

23 
Harlan Laboratories Study C31 043 

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Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

CONTENTS 

Report 

Study Plan Page2 

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

SIGNATURES ................................................................................................................................ 3 

PREFACE ....................................................................................................................................... 4 

General Information ............................................................................................................... 4 

Responsibilities ...................................................................................................................... 4 

Schedule ................................................................................................................................. 4 

Good Laboratory Practice ...................................................................................................... 5 

Data Requirements I Test Guidelines .................................................................................... 5 

Animal Welfare ...................................................................................................................... 5 

Amendment and Deviation Procedures ................................................................................. 5 

Archiving ............................................................................................................................... 6 

1 PURPOSE .............................................................................................................................. 7 

2 MATERIALS AND METHODS ........................................................................................... 8 

2.1 Test System .............................................................................................................. 8 

2.2 Allocation ................................................................................................................ 9 

2.3 Husbandry ................................................................................................................ 9 

2.4 Test Item and Vehicle ............................................................................................ lO 

2.4.1 Test Item .................................................................................................. lO 

2.4.2 Vehicle ..................................................................................................... lO 

2.5 Preparation of Dose Formulations ......................................................................... ll 

2.6 Test Item Administration ....................................................................................... ll 

2.7 Rationale ................................................................................................................ ll 

2.8 Observations .......................................................................................................... 12 

2.9 Pathology ............................................................................................................... l2 

2.9.1 Necropsy .................................................................................................. l2 

2.10 Determination and Scoring of Clinical Signs ........................................................ 12 

2.10.1 Observation .............................................................................................. l2 

2.11 Statistical Analysis ................................................................................................ 13 

2.12 Data Compilation ................................................................................................... 13 

2.13 Test Procedure: Guideline Excerpt... ..................................................................... l4 

3 REPORTING ....................................................................................................................... 15 

4 DISTRIBUTION ................................................................................................................. 15 

09009e53806cb39a 24 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black 1 

Harlan Laboratories Study C31043 

Leuco Sulfur Black I 

SIGNATURES 

Study Director: 

Management: 

Quality Assurance: 

For the Sponsor: 

09009e53806cb39a 

Report 

Study Plan 

Dr. C. Simon 

CJ ( 'cs~------­ D~~~:·;a··~·;p~;,···~-z~· 

{{r-u) Dr. H. Fankhauser 

.......... ~.q!.~~~~ ........ . Date: "?.. o -JAiv --Wos 

S. van Dongen 

Dr. Thomas Roth 

SCCGmbH 

Page 3 

25 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black l 

Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Laboratoryffechnical Coordinator: 

Quality Assurance: 

HeadofQA: 

Schedule 

Anticipated Experimental Starting 

Date: 

Delivery of animals (first dose): 

Acclimatization (first dose): 

Administration I Treatment: 

Termination (Necropsy): 

09009e53806cb39a 

Report 

Study Plan 

Leuco Sulfur Black 1 

Acute Oral Toxicity Study in Rats 

Clariant Produkte (Schweiz) AG 

Rothausstrasse 61 

CH-4132 Muttenz I Switzerland 

Dr. Thomas Roth 

SCCGmbH 

Mikroforum Ring 1 

D-55234 W endelsheim /Germany 

Harlan Laboratories Ltd. 

Wolferstrasse 4 

4414 Fullinsdorf I Switzerland 

Harlan Laboratories Ltd. 

Quality Assurance GLP 

Zelgliweg 1 

4452 Itingen I Switzerland 

Dr. C. Simon 

G. Arcelin 

C. Weng 

T.Fink 

20-Jan-2009 

20-Jan-2009 

20-Jan-2009 to 26-Jan-2009 

27-Jan-2009 

At least 14 days after administration 

Page4 

26 
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Report 

Study Plan 

Anticipated Experimental Completion At last necropsy 

Date: 

Anticipated Date of Draft Report: 03-Mar-2009 (depending on the number of dose 

levels) 

Good Laboratory Practice 

Page 5 

This study will be performed in compliance with the Swiss Ordinance relating to Good 

Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the 

OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 

1997 by decision ofthe OECD Council [C (97)186/Final]. 

Data Requirements I Test Guidelines 

This study will follow the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

• OECD Guidelines for the Testing of Chemicals, Number 423 "Acute Oral ToxicityAcute Toxic Class Method", adopted 17th December 2001. 

• Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods 

pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the 

Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals 

(REACH), B. I tris. ACUTE ORAL TOXICITY - ACUTE TOXIC CLASS METHOD 

(Official Journal No L 142,31105/2008 p. 0158-0173). 

Animal Welfare 

This study will be performed in an AAALAC-accredited laboratory in accordance with the Swiss 

Animal Protection Law under license no. 34. 

Amendment and Deviation Procedures 

Amendments (planned changes) to the study plan will be issued and signed by the Study Director 

and will become effective at the time of Study Director signature. The Sponsor will receive the 

original amendment, which must be signed and returned to Harlan Laboratories Ltd., and one 

copy. The amendment will be distributed (see Distribution) and added to all copies of the study 

plan. 

Deviations (unplanned changes) to the study plan will be documented and acknowledged by the 

Study Director and maintained with the raw data. The report will reflect any deviations. The 

Sponsor will be promptly informed of any relevant deviations from the study plan. 

09009e53806cb39a 27 
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Archiving 

Report 

Study Plan Page 6 

Harlan Laboratories Ltd. ( 4452 Itingen/Switzerland), will retain the study plan, raw data, a 

sample of the test.item and the final report of the present study for at least ten years. No data will 

be discarded without the Sponsor's written consent. 

09009e53806cb39a 28 
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1 PURPOSE 

Report 

Study Plan Page 7 

The purpose of this study is to assess the acute toxicity of Leuco Sulfur Black 1 when 

administered by a single oral gavage to rats, followed by an observation period of 14 days or 

more, depending on the occurrence of clinical signs of toxicity. 

This study should provide information for both hazard assessment and for hazard classification 

purposes. 

09009e53806cb39a 29 
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Harlan Laboratories Study C31043 

Leuco Sulfur Black I 

Report 

Study Plan Page 8 

2 MATERIALS AND METHODS 

General Remark 

Details of the materials and methods that are not specified in the subsequent sections of this 

study plan are contained in the appropriate Harlan Laboratories Ltd. standard operating 

procedures. 

2.1 Test System 

Animals: Rat, HanRcc: WIST(SPF) 

Rationale: 

Breeder: 

Number of Animals: 

Age (when treated): 

Body Weight Range (when treated): 

Identification: 

Randomization: 

Acclimatization: 

09009e53806cb39a 

Recognized by international guidelines as a 

recommended test system. 

Harlan Laboratories Ltd. 

Laboratory Animal Services 

Wolferstrasse 4 

4414 Fullinsdorf I Switzerland 

At least 3 females 

Per step three animals of a single sex (normally 

females) are used. When the test is conducted in 

males, adequate justification should be provided. 

Approximately 8- 12 weeks 

Approximately 200 g ± 20 % 

Unique cage number and corresponding color-coded 

spots on the tail. The animals will be marked at 

acclimatization start. 

Selected by hand at time of delivery. 

No computer generated randomization program. 

Under laboratory conditions, after health 

examination. Only animals without any visible signs 

of illness will be used for the study. 

30 
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2.2 Allocation 

Report 

Study Plan Page 9 

The animals will be distributed as follows: 

Dose 

Levels: Group: Animal Nos.: 

2000 mg!kg 1-3 

The further dose levels and animal numbers will be specified in the raw data and report. 

2.3 Husbandry 

Room Numbers: 

Conditions: 

Accommodation: 

Diet: 

Water: 

09009e53806cb39a 

Will be specified in the raw data and the report 

(Fullinsdorf). 

Standard laboratory conditions. The animal room 

will be air-conditioned with 10-15 air changes per 

hour. The air will be continuously monitored for 

temperature and relative humidity. The ranges for 

room temperature and relative humidity will be 22 ± 

3 oc and 30-70 %, respectively, although the upper 

range for humidity may be exceeded during room 

cleaning. The animals will be provided with an 

automatically controlled light cycle of 12 hours light 

and 12 hours dark. Music will be played during the 

daytime light period. 

In groups of three in Makrolon type-4 cages with 

wire mesh tops and standard softwood bedding 

('Lignocel' Schill AG, CH-4132 

Muttenz/Switzerland). 

Pelleted standard Provimi Kliba 3433 rat/mouse 

maintenance diet (Provimi Kliba AG, CH-4303 

Kaiseraugst/ Switzerland) ad libitum (except for the 

overnight fasting period prior to intubation and 

approximately 3-4 hours post dose). Results of 

analyses for contaminants will be archived at Harlan 

Laboratories Ltd. 

Community tap-water, from Fullinsdorf ad libitum. 

Results of bacteriological, chemical and contaminant 

analyses will be archived at Harlan Laboratories Ltd. 

31 
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2.4 Test Item and Vehicle 

2.4.1 Test Item 

Report 

Study Plan Page 10 

The following information was provided by the sponsor: 

Identification: 

Description: 

Batch Number: 

Composotion: 

Stability of Test Item: 

Expiry Date: 

Stability of Test Item Dilution: 

Storage Conditions: 

Safety Precautions: 

2.4.2 Vehicle 

Leuco Sulfur Black l 

Paste I liquid 

ESA0007595, 11.07.08 

Leuco Sulfur Black 1: 

2,4-Dinitrochlorobenzene: 

2,4-Dinitrophenole: 

Sulfate: 

Chloride: 

Sodium: 

Water: 

Stable under storage conditions. 

11-Jul-2011 

> 4h in water. 

45.5% (w/w) 

<20ppm 

<20ppm 

970ppm 

750ppm 

1.3% (w/w) 

53% (w/w) 

At room temperature (range of 20 ± 5 °C), light 

protected. 

Routine hygienic procedures will be used to ensure 

the health and safety of the personnel. 

Purified water was prepared at Harlan Laboratories Ltd. (deionised water which was processed 

and treated by the PURELAB Option-R unit. This latter links four purification technologies: 

reverse osmosis, adsorption, ion-exchange and photo oxidation). 

The vehicle was chosen after a non-GLP solubility trial which was performed before the study 

initiation date. 

09009e53806cb39a 32 
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Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

Report 

Study Plan 

2.5 Preparation of Dose Formulations 

Page II 

The dose levels are in terms of the active ingredient (Leuco Sulfur Black 1; 45.5% w/w) in 

the test item. 

The dose formulations will be prepared shortly before each dosing occasion. 

The test item will be weighed into a tared glass beaker on a suitable precision balance and the 

vehicle will be added (weight:volume). The formulations will be prepared using a suitable 

homogenizer. 

Homogeneity of the test item in the vehicle will be maintained during administration using a 

magnetic stirrer. 

2.6 Test Item Administration 

The animals will receive a single dose of the test item by oral gavage administration after being 

fasted for approximately 16 to 20 hours, but with free access to water. Food will be presented 

approximately 3 to 4 hours after dosing. 

Dosing will start in three female animals at a dosage level of 2000 mg/kg active ingredient 

(corresponding to 4396 mglkg of the test item as delivered; based on the amount of 45.5% (w/w) 

Leuco Sulfur Black 1 ). The test procedure will follow the attached schema described under 

Section 2.13 (Test Procedure with a Starting Dose of 2000 mg/kg Body Weight which is based 

on Annex 2d, OECD Guideline 423, adopted 17th December 2001) unless otherwise specified by 

the sponsor. 

The sponsor will be informed prior to each new dosing phase. 

If an additional dose level (or levels) is necessary, additional naive animals will be used. This 

will be described in the raw data and report. 

The application volume will be 10 mL/kg body weight. 

Remark: Animals killed for humane reasons are considered in the same way as animals that die 

on test. 

2. 7 Rationale 

Oral administration is considered to be an appropriate application method as it is a possible route 

of human exposure during manufacture, handling and use of the test item. 

09009e53806cb39a 33 
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Harlan Laboratories Study C31 043 

Leuco Sulfur Black I 

2.8 Observations 

Report 

Study Plan Page 12 

The following observations will be recorded as follows: 

Viability I Mortality: 

Clinical Signs: 

Body Weights: 

2.9 Pathology 

2.9.1 Necropsy 

Daily during the acclimatization period, during the 

first 30 minutes and at approximately I, 2, 3 and 5 

hours after administration on test day I (in common 

with the clinical signs) and twice daily during days 

2-15. 

Daily during the acclimatization period, during the 

first 30 minutes and at approximately I, 2, 3 and 5 

hours after administration on test day I, depending 

on the occurrence of clinical signs of toxicity. Once 

daily during days 2-15. 

On test days I (prior to administration), 8 and 15. 

All surviving animals will be killed at the end of the observation period by intraperitoneal 

injection of Pentobarbitone at a dose of at least 2.0 mL/kg body weight (324 mg sodium 

pentobarbitone/kg body weight) or by carbon dioxide asphyxiation and discarded after 

macroscopic abnormalities have been recorded. The method used will be described in the raw 

data and report. 

Animals which die spontaneously during the observation period will be necropsied as soon as 

they are found dead and any abnormalities recorded. 

No organs or tissues will be retained. 

Note: All animals that exhibit signs of undue stress or discomfort as judged by the study 

director and the technical coordinator will be sacrificed immediately for ethical reasons. The 

sponsor will be informed immediately. The reason for sacrifice will be included in the final 

report. 

2.10 Determination and Scoring of Clinical Signs 

2.10.1 Observation 

Data will be summarized in tabular form, showing for each individual animal the clinical signs at 

each measurement interval. All findings will be described. 

09009e53806cb39a 34 
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Harlan Laboratories Study C31043 

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2.11 Statistical Analysis 

No statistical analysis will be used. 

2.12 Data Compilation 

Body weights will be recorded on-line. 

Report 

Study Plan 

Clinical signs will be recorded on data sheets. 

Page 13 

Mortality/viability will be compiled into the RCC Tox Computer System during recording and/ 

or recorded on data sheets. 

Macroscopic findings will be compiled into the RCC Tox Computer System during recording. 

The RCC Tox Computer System (RCC-Tox-Lims) has been validated with respect to data 

collection, storage and retrievability. 

If technical problems arise during the on-line recording/immediate compilation of data during 

observation or additional registration of unplanned data, recording will be carried out on data 

sheets. These data will be transcribed later for compilation and analysis. 

09009e53806cb39a 35 
§ 

"' "' "" 

" 

rE 

w 

0'1 

GHS 

LD50 cut-oft 

mg.1<gb.w. 

OECD/OCDE 

ANNEX 2d: TEST PROCEDURE WITH A STARTING DOSE OF 2000 MG/KG BODY WEIGHT 

-per step thflt animals of a single aex ( normally ltmalts ) are used 

-0,1.2,3: Numberofmorlbund ordeJdanlmalt at 11ch step 

• GHS: GloboHy Harmonized CloaoltlcoUon Sylllem (mg/kg b.w.) 

423 
Harlan Laboratories Study C31 043 

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3 REPORTING 

Report 

Study Plan Page 15 

A GLP-compliant draft report will be submitted to the study monitor for scientific review. 

Following receipt of the sponsor's comments, a QA-audited final report will be issued. 

4 DISTRIBUTION 

This study plan will be distributed as follows: 

Sponsor 

Study Monitor (responsible for distribution 

to the Sponsor company): l x (copy) 

Harlan Laboratories Ltd. 

Study File: 

Study Director: 

Deputy Study Director: 

Laboratory Technical Coordinator: 

Logistics: 

QA: 

09009e53806cb39a 

l x (original) 

l x (copy) 

l x (copy) 

l x (copy) 

l x (copy) 

l x (copy) 

37 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black 1 

Report 

09009e53806cb3a2 

FIRST AMENDMENT TO STUDY PLAN 

LEUCO SULFUR BLACK 1 

Acute Oral Toxicity Study in Rats 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

Version: 

Dr. C. Simon 

Harlan Laboratories Ltd. 

Wolferstrasse 4 

4414 Fullinsdorf I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1/ Switzerland 

Harlan Laboratories Study C31043 

Final 

Page 1 of9 

38 
Harlan Laboratories Study C31 043 

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Harlan Laboratories Study C31043 

Leuco Sulfur black I 

SIGNATURES 

Study Director on signature of 

this amendment to study plan: 

Management: 

For Sponsor: 

09009e53806cb3a2 

Report 

First Amendment to Study Plan Page 2 

G.7 

.. /o. ....... :.i.~ ... 

Date:/!'}- ~~ ~o5 

Dr. H. Fankhauser 

~ ...... k .. ~ .. 

nate: 0, . {lV _ "2-<!J&fJ 

39 
Harlan Laboratories Study C31 043 

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Leuco Sulfur black I 

PAGE 

4 

CONCERNING 

Responsibilities 

PRESENT 

Study Director: 

Deputy Study Director: 

( ... ) 

NEW 

Study Director: 

Deputy Study Director: 

( ... ) 

Report 

First Amendment to Study Plan 

Dr. C. Simon 

G. Arcelin 

Dr. C. Simon until 08-Jul-2009 

G. Arcelin from 09-Jul-2009 

G. Arcelin until 08-Jul-2009 

E. Rached from 09-Jul-2009 

REASON FOR ALTERATION 

Page 3 

New internal organization. Since Dr. C. Simon has left the department, G. Arcelin was 

nominated as Study Director to finalize the report. Therefore, a new Deputy of Study Director 

took place. 

09009e53806cb3a2 40 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black 1 

Report 

Harlan Laboratories Study C31043 

Leuco Sulfur black I 

First Amendment to Study Plan 

PAGE 

9 

CONCERNING 

2.2 Allocation 

PRESENT 

The animals will be distributed as follows: 

Dose 

Levels: Group: Animal Nos.: 

2000 mglkg 1-3 

The further dose levels and animal numbers will be specified in the raw data and report. 

NEW 

The animals will be distributed as follows: 

Dose 

Levels: 

2319 mg active ingredient I kg 

(4396 mg test item I kg) 

Group: Animal Nos.: 

1-3 

The further dose levels and animal numbers will be specified in the raw data and report. 

REASON FOR THE ALTERATION 

Reanalysis of the test item by the sponsor resulted in new content of active ingredient. 

09009e53806cb3a2 

Page4 

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PAGE 

10 

CONCERNING 

2.4.1 Test Item 

PRESENT 

Report 

First Amendment to Study Plan Page 5 

The following information was provided by the sponsor: 

Identification: 

Description: 

Batch Number: 

Composition: 

Stability of Test Item: 

Expiry Date: 

Stability of Test Item Dilution: 

Storage Conditions: 

Safety Precautions: 

09009e53806cb3a2 

Leuco Sulfur Black 1 

Paste /liquid 

ESA0007595, 11.07.08 

Leuco Sulfur Black 1: 

2,4-Dinitrochlorobenzene: 

2,4-Dinitrophenole: 

Sulfate: 

Chloride: 

Sodium: 

Water: 

Stable under storage conditions. 

11-Jul-2011 

> 4h in water. 

45.5% (w/w) 

<20ppm 

<20ppm 

970ppm 

750ppm 

1.3% (w/w) 

53% (w/w) 

At room temperature (range of 20 ± 5 °C), light 

protected. 

Routine hygienic procedures will be used to ensure 

the health and safety of the personnel. 

42 
Harlan Laboratories Study C31 043 

Leuco Sulfur Black 1 

Harlan Laboratories Study C31 043 

Leuco Sulfur black I 

NEW 

Report 

First Amendment to Study Plan Page 6 

The following information was provided by the sponsor: 

Identification: 

CAS No.: 

Description: 

Batch Number: 

Composition: 

Stability of Test Item: 

Expiry Date: 

Stability of Test Item Dilution: 

Storage Conditions: 

Safety Precautions: 

Leuco Sulfur Black 1 

66241-11-0 

Thixothrophic, black, wet paste 

ESA0007595 

Leuco Sulfur Black 1 

(calculated by amount of bycompounds): 

2,4-Dinitrochlorobenzene: 

2,4-Dinitrophenole: 

Sulfate: 

Chloride: 

Sodium: 

Water (by Karl-Fischer 

titration): 

Stable under storage conditions. 

11-Jul-2011 

> 4h in water. 

52.75% (w/w) 

<20ppm 

<20ppm 

970ppm 

750 ppm 

1.3% (w/w) 

45.75% (w/w) 

At room temperature (range of 20 ± 5 °C), light 

protected. 

Routine hygienic procedures will be used to ensure 

the health and safety of the personnel. 

REASON FOR THE ALTERATION 

A new Certificate of Analysis was provided by the sponsor after a reanalysis of the test item. 

09009e53806cb3a2 43 
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Harlan Laboratories Study C31 043 

Leuco Sulfur black I 

PAGE 

II 

CONCERNING 

Report 

First Amendment to Study Plan 

2.5 Preparation of Dose Formulations 

PRESENT 

Page 7 

The dose levels are in terms of the active ingredient (Leuco Sulfur Black I; 45.5% w/w) in the 

test item. 

( ... ) 

NEW 

The dose levels are in terms of the active ingredient (Leuco Sulfur Black I; 52.75% w/w) in the 

test item. 

( ... ) 

REASON FOR THE ALTERATION 

Reanalysis of the test item by the sponsor resulted in new content of active ingredient. 

09009e53806cb3a2 44 
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Harlan Laboratories Study C31 043 

Leuco Sulfur black I 

PAGE 

11 

CONCERNING 

2.6 Test Item Administration 

PRESENT 

( ... ) 

Report 

First Amendment to Study Plan Page 8 

Dosing will start in three female animals at a dosage level of 2000 mg/kg active ingredient 

(corresponding to 4396 mglkg of the test item as delivered; based on the amount of 45.5% (w/w) 

Leuco Sulfur Black 1 ). The test procedure will follow the attached schema described under 

Section 2.13 (Test Procedure with a Starting Dose of 2000 mg/kg Body Weight which is based 

on Annex 2d, OECD Guideline 423, adopted 17th December 2001) unless otherwise specified by 

the sponsor. 

( ... ) 

NEW 

( ... ) 

Dosing will start in three female animals at a dosage level of 2319 mg/kg active ingredient 

(corresponding to 4396 mg/kg of the test item as delivered; based on the amount of 52.75% 

(w/w) Leuco Sulfur Black 1). The test procedure will follow the attached schema described 

under Section 2.13 (Test Procedure with a Starting Dose of 2000 mg/kg Body Weight which is 

based on Annex 2d, OECD Guideline 423, adopted 17th December 2001) unless otherwise 

specified by the sponsor. 

( ... ) 

REASON FOR THE ALTERATION 

Reanalysis of the test item by the sponsor resulted in new content of active ingredient. 

09009e53806cb3a2 45 
Harlan Laboratories Study C3I 043 

Leuco Sulfur Black I 

Harlan Laboratories Study C31 043 

Leuco Sulfur black I 

DISTRIBUTION 

Report 

First Amendment to Study Plan 

This amendment will be distributed as follows: 

Sponsor 

Study Monitor (responsible for distribution 

to the Sponsor company): I x (copy) 

Harlan Laboratories Ltd. 

Study File: 

Study Director: 

Deputy Study Director: 

Laboratory Technical Coordinator: 

Logistics 

QA: 

I x (original) 

I x (copy) 

I x (copy) 

I x (copy) 

I x (copy) 

I x (copy) 

180 ON SNI\7'1NOO 

09009e53806cb3a2 

Page 9 

46