# REPORT 

# LEUCO SULFURBLACK 1



# Acute Oral Toxicity Study in Rats

Study Director: Test Facility:

Sponsor: G. Arcelin Harlan Laboratories Ltd. Wolferstrasse 4 4414 FiillinsdorfI 

### 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 of46



# CONTAINS NO CBI 

# harlanTM


# ....

### Harlan Laboratories Study C31043 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 3.10 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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### 2Harlan Laboratories Study C31043 Leuco Sulfur Black 1 Report


### Study 

### Plan .................................................................................................................... 23 First Amendment to Study 

### Plan ................................................................................... 38

09009e53&06cb3ab 

### 3

### Harlan Laboratories Study C31043 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 ofGood 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 ofthe data. Study Director:

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### 4

### Harlan Laboratories Study C31043 Leuco Sulfur Black 1 

### Report 

# QUALITYASSURANCE 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 ofReports to Dates and Types ofQA Inspections the StudyDirector 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 

### 5Harlan Laboratories Study C31 043 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 FiillinsdorfI 

### 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



### 6Harlan Laboratories Study C31043 Leuco Sulfur Black I 

## Schedule Experimental Starting Date: Termination (Necropsy): Delivery ofAnimals: 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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### 7Harlan Laboratories Study C31 043 Leuco Sulfur Black I 

# 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 of2319 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 ofthe animals was within the range commonly recorded for this strain and age. No macroscopic findings were observed at necropsy. The median lethal dose ofLeuco 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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### 8Harlan Laboratories Study C31043 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.

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### 9Harlan Laboratories Study C31043 Leuco Sulfur Black I Report 

# 3 MATERIALS AND METHODS 3.1 Test System Animals: Rationale: Breeder:
Number ofAnimals per Group: Total Number ofAnimals: 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 ofdelivery. No computer generated randomization program. Under laboratory conditions, after health examination. Only animals without any visible signs ofillness 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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### 10Harlan Laboratories Study C31043 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 ofbacteriological, 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:

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### Leuco Sulfur Black 

### 1 66241-11-0 Thixothrophic, black, wet paste ESA0007595 Leuco Sulfur Black 1 (calculated by amount ofbycompounds): 2,4-Dinitrochlorobenzene: 2,4-Dinitrophenole: Sulfate: Chloride: 52.75% (wlw) <20ppm <20 ppm 970 ppm 750 ppm 11Harlan Laboratories Study C31043 Leuco Sulfur Black I



### Stability ofTest Item: Expiry Date: Stability ofTest 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 ofthe 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 ofcompliance. 

## 3.5 Preparation ofDose Formulations The dose levels are in terms ofthe 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.

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### 12Harlan Laboratories Study C31043 Leuco Sulfur Black I 

## 3.6 Test Item Administration Report



### The animals received a single dose ofthe test item by oral gavage administration at 2319 mglkg body weight active ingredient (corresponding to 4396 mg/kg ofthe test item delivered; based on the amount of52.75% (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 ofhuman 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 oftoxicity. Once daily during days 2-15. On test days 1 (prior to administration), 8 and 15.

All animals were killed at the end ofthe 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.

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### 13Harlan Laboratories Study C31043 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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### 14

### Harlan Laboratories Study C31043 Leuco Sulfur Black 1 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 ofthe animals on test 1. 

### On test day 2, all three females were noted with black-stained feces and the cage ofthe 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 ofclinical 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 ofany clinical signs from test day 6 until the end ofthe 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.

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### 15Harlan Laboratories Study C31043 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)

16Harlan Laboratories Study C31043 Leuco Sulfur Black I



# 6 INDIVIDUAL TABLES

09009e53806d5407 Report



### 17Harlan Laboratories Study C31043 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* ..} ..} 

### ..} ..} ..} 

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### 6 7 8 9 10 11 12 13 ..} ..} ..} 

### ..} ..} v ..} ..} 

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## *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.

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### 18

### Harlan Laboratories Study C3l043 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

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### 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



### 19Harlan Laboratories Study C31043 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 

### 20Harlan Laboratories Study C31043 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

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### 21

### Harlan Laboratories Study C31043 Leuco Sulfur Black I Report



# APPENDIX I -STUDY PLAN AND AMENDMENT

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## 22Harlan Laboratories Study C31043 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 FullinsdorfI Switzerland 

### Clariant Products (Switzerland) Ltd. Rothausstrasse 61 CH-4132 Muttenz 1/ Switzerland
Harlan Laboratories Study C31043 Final
Page 1 of 15



### 23Harlan Laboratories Study C31043 Leuco Sulfur Black I

Harlan Laboratories Study C31043 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 ofDose 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 ofClinical 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

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### 24Harlan Laboratories Study C31043 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



### 25Harlan Laboratories Study C31043 Leuco Sulfur Black l

Harlan Laboratories Study C31043 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 ofanimals (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 Wendelsheim /Germany Harlan Laboratories Ltd. Wolferstrasse 4 4414 FullinsdorfI 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



### 26Harlan Laboratories Study C31043 Leuco Sulfur Black 1

Harlan Laboratories Study C31043 Leuco Sulfur Black I Report

Study Plan Anticipated Experimental Completion At last necropsy Date: Anticipated Date ofDraft Report: 03-Mar-2009 (depending on the number ofdose 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 ofthe 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 ofany relevant deviations from the study plan.

09009e53806cb39a 

### 27Harlan Laboratories Study C31043 Leuco Sulfur Black 1

Harlan Laboratories Study C31043 Leuco Sulfur Black I 

### Archiving Report

Study Plan Page 6

Harlan Laboratories Ltd. (4452 Itingen/Switzerland), will retain the study plan, raw data, a sample ofthe test.item and the final report ofthe present study for at least ten years. No data will be discarded without the Sponsor's written consent.

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### 28Harlan Laboratories Study C31043 Leuco Sulfur Black I

Harlan Laboratories Study C31043 Leuco Sulfur Black I 

## 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 ofclinical signs oftoxicity. This study should provide information for both hazard assessment and for hazard classification purposes.

09009e53806cb39a 

### 29Harlan Laboratories Study C31043 Leuco Sulfur Black I

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 ofAnimals: 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 ofa single sex (normally females) are used. When the test is conducted in males, adequatejustification 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 ofdelivery. No computer generated randomization program. Under laboratory conditions, after health examination. Only animals without any visible signs ofillness will be used for the study.



### 30Harlan Laboratories Study C31043 Leuco Sulfur Black 1

Harlan Laboratories Study C31043 Leuco Sulfur Black I 

### 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 ofbacteriological, chemical and contaminant analyses will be archived at Harlan Laboratories Ltd.



### 31Harlan Laboratories Study C3l043 Leuco Sulfur Black I

Harlan Laboratories Study C31043 Leuco Sulfur Black I 

### 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 ofTest Item: Expiry Date: Stability ofTest 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 of20 ± 5 °C), light protected. Routine hygienic procedures will be used to ensure the health and safety ofthe 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 

### 32Harlan Laboratories Study C31043 Leuco Sulfur Black I

Harlan Laboratories Study C31043 Leuco Sulfur Black I Report

Study Plan 

### 2.5 Preparation ofDose 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 ofthe 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 ofthe test item as delivered; based on the amount of45.5% (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 ofhuman exposure during manufacture, handling and use ofthe test item.

09009e53806cb39a 

### 33Harlan Laboratories Study C31043 Leuco Sulfur Black I

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 ofclinical signs oftoxicity. 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 ofClinical 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 

### 34Harlan Laboratories Study C31043 Leuco Sulfur Black I

Harlan Laboratories Study C31043 Leuco Sulfur Black I 

### 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§"'"'"""rEw0'1GHSLD50cut-oftmg.1<gb.w.OECD/OCDEANNEX2d:TESTPROCEDUREWITHASTARTINGDOSEOF2000MG/KGBODYWEIGHT-perstepthfltanimalsofasingleaex(normallyltmalts)areused-0,1.2,3:NumberofmorlbundordeJdanlmaltat11chstep•GHS:GloboHyHarmonizedCloaoltlcoUonSylllem(mg/kgb.w.)423....--3"'...."'d..,=n=-=..,=...=--...=t'!'jil'in..,"CC....t""'::t0>c::e;8§t'"'::C[J)2c::t""'"-§::0"'00::>....,0....,0::t'"'t:C-"'Z'<::r0....,:>I"o·-'J>[J)2-ooQ.'<8'n...,-("').....0+>-...,.....00g"tlg?;"0§.i-1>Harlan Laboratories Study C31043 Leuco Sulfur Black l

Harlan Laboratories Study C31043 Leuco Sulfur Black I 

## 3 REPORTING Report

Study Plan Page 15

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



### 37Harlan Laboratories Study C31043 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 FullinsdorfI Switzerland 

### Clariant Products (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1/ Switzerland Harlan Laboratories Study C31043 Final
Page 1 of9



### 38

### Harlan Laboratories Study C31043 Leuco Sulfur Black 

### 1

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

39Harlan Laboratories Study C31043 Leuco Sulfur Black 1

Harlan Laboratories Study C31043 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 FORALTERATION 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 

### 40Harlan Laboratories Study C31043 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 FORTHE ALTERATION Reanalysis ofthe test item by the sponsor resulted in new content ofactive ingredient.

09009e53806cb3a2 Page4



### 41Harlan Laboratories Study C31043 Leuco Sulfur Black 1

Harlan Laboratories Study C31043 Leuco Sulfurblack I 

## 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 ofTest Item: Expiry Date: Stability ofTest 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 of20 ± 5 °C), light protected. Routine hygienic procedures will be used to ensure the health and safety ofthe personnel.



### 42Harlan Laboratories Study C31043 Leuco Sulfur Black 1

Harlan Laboratories Study C31043 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 ofTest Item: Expiry Date: Stability ofTest 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 ofbycompounds): 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 of20 ± 5 °C), light protected. Routine hygienic procedures will be used to ensure the health and safety ofthe personnel. 

## REASON FORTHE ALTERATION A new Certificate ofAnalysis was provided by the sponsor after a reanalysis ofthe test item.

09009e53806cb3a2 

### 43Harlan Laboratories Study C31043 Leuco Sulfur Black I

Harlan Laboratories Study C31043 Leuco Sulfurblack I 

## PAGE II 

## CONCERNING Report

First Amendment to Study Plan

2.5 Preparation ofDose Formulations 

## PRESENT Page 7

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

### ...) 

## NEW The dose levels are in terms ofthe active ingredient (Leuco Sulfur Black I; 52.75% w/w) in the test item. (

### ...) 

## REASON FOR THEALTERATION Reanalysis ofthe test item by the sponsor resulted in new content ofactive ingredient.

09009e53806cb3a2 

## 44Harlan Laboratories Study C31043 Leuco Sulfur Black l

Harlan Laboratories Study C31043 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 ofthe test item as delivered; based on the amount of45.5% (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 FORTHE ALTERATION Reanalysis ofthe test item by the sponsor resulted in new content ofactive ingredient.

09009e53806cb3a2 

### 45Harlan Laboratories Study C3I043 Leuco Sulfur Black I

Harlan Laboratories Study C31043 Leuco Sulfurblack 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