DISCLAIMER 

This report is the property of Clariant. 

Its content is confidential and not for general 

release. It is not allowed to cite and reveal the report or its 

content to a third party without prior written approval of 

Clariant. 

REPORT 

31-15153 

haria nTh .... 

Leuco Sulfur Black 1: Determination of the Water Solubility 

Data Requirements I 

Test Guidelines: 

Study Director: 

Test Facility: 

Sponsor: 

OECDNo.l05 

EEC Directive 92/69, A.6 

Council Regulation (EC) 44012008, A.6 

Dr Martin Kiihne 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 Itingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Study Identification: Harlan Laboratories Study C75560 

Version: Final 

Study Completion Date: March 11, 2010 

Page 1 of 16 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

CONTENTS 

Report Page2 

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

LIST OF TABLES .......................................................................................................................... 2 

GLP CERTIFICATE ....................................................................................................................... 3 

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

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

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

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

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

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

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

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

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

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

3 MATERIALS AND METHODS ......................................................................................... 10 

3.1 Test Item ................................................................................................................ 10 

3.2 Test System ............................................................................................................ 10 

3.3 Reagents and Apparatus ........................................................................................ 11 

3.4 Principle ................................................................................................................. 11 

3.5 Performance of the Test. ........................................................................................ 12 

3.6 Analytical Method ................................................................................................. 12 

3.6.1 System Suitability check ......................................................................... 12 

3.7 Data Handling ........................................................................................................ 13 

4 RESULTS AND DISCUSSION .......................................................................................... 14 

5 CONCLUSION .................................................................................................................... 15 

6 TABLES .............................................................................................................................. 16 

LIST OF TABLES 

Table 1 Data of the Performance of the Water Solubility Test... ............................................. 16 

Table 2 Results of the Water Solubility of Leuco Sulfur Black 1 ........................................... 16 

-~v·1( 

V~t·!·~ .ioJ~ ~1 '¥· 

·.' 
Harlan Laboratories Study C75560 

Leuco Sulfur Black 1 

GLP CERTIFICATE 

" S<-oscho !ill-scllall V CoftfHHatioft wiue ConfHif.UiOM S•tn•• 

Conftdlf.,_. mu• 

Report 

,..._ ..... __ Ftdltll __ DtotnNnt of .._, Aff~t~~ DtotA ., __ _ ftoftt•l hptttfhtftt of tM ifmfOftmtQI. 

Tro-'- !MftY .... C-OETIC 

------ SWlSSme ~ d" ..... _ ... __ 

JC 

Statement of GLP Compliance 

Acoonling to Miele 14 paragraph 3 Ordinance on Good Laboratory Pradlce (OGLP. SR 813.112.11 

The nolillcltion IIUihorily for chemicals confilml that till following test facility was inspected with 

respect to till complilnce with till SwisS Ordinance on Good L.aborllory Pradlce, adopted on 18th 

May 2005 [OGLP. SR 813.112.11. This Ordinance is based on till oeco Principles of Good 

Laboralary Practice, as reviled in 1997 and adopt8d on 26th November 1997 by decision of lhe 

OECD Council [C(97)1861finaq. 

Unequivocal name ll1d add,_ 

of till lilt facility: 

Harlan Llboratorles Ltd. 

Zelgliweg 1 

4452 ltingen, Switzelllnd. 

Atus of expertise ICCOrding to 

lfllcle 3 par~graph 1 leu. d OGLP: 

1 J Physicak:hernlcal testing, 

2J T oxldty studies, 

41 Environmental toxicity studies on aquatic 

and terrestrial organilms, 

51 Studies on behaviour in water. 101 and air; 

bioaccumulltlon, 

61 Relldue studies, 

7 .I Studies on etr1c:1s on I'II8IIOCOIIIW and 

naturll ecosylllml, 

61 Analytic:IIIIICI clinical c:hemist1y tedng, 

9./ Other studies (ur.ty pl\8miiCCIIclgy and 

animll melabollsm). 

Inspection authority: Federal Olllce for till Environment (FOEN) I Federal Olllce of Public Heelltl 

(FOPH) I Swiss AtJency for Thenlpeutic Products (Swiarnedic) 

Date of inspection: 05th to 09lh and 26th to 30th November 2007 

Date of decision: 30th Aplil2008 

Based on the above mentionad decision it can be confirmed that till above mentioned test facitity 

is able to conduct studies acc:ording to the aforementioned area of expertise in compliance wilh 

the principles of GLP. The above mentioned test facility is listed In till register and GLP list 

acc:ording to the Miele 14 OGLP and is inspected on a regular basis according to Article 6 

paragraph 2 OGLP. 

TN~.....,_totCIWII-• _,_OIIoo., .. 

___ ~INllilliiiiDn.._.,.., c-____ ...... ....., ..... IOL")IIII' .. ,OIN. .. ,OPH ... ......._ ,.,._c_._ 

_ ... ,,...,.._ ... ,topt3ZJ7)1!,f_ .. ,.,3ZI54. 

Page 3 
Harlan Laboratories Study C75560 

Leuco Sulfur Black 1 

GOOD LABORATORY PRACTICE 

Report 

STATEMENT OF COMPLIANCE 

Harlan Laboratories Study: 

Test Item: 

Study Director: 

C75560 

Leuco Sulfur Black I 

Dr Martin Kiihne 

Page4 

Study Title: Leuco Sulfur Black 1: Determination of the Water 

Solubility 

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 on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. 

These principles are compatible with Good Laboratory Practice regulations specified by 

regulatory authorities throughout the European Community, the United States (EPA and FDA), 

and Japan (MHW, MAFF and MET!). 

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

Study Director: Dr Martin Kiihne 

_/fA {_;;_~ ...... ~.::.:.: ...................... .. 

Date: (1 ~.: ( AA 2 o 10 

I 
Harlan Laboratories Study C75560 

Leuco Sulfur Black l 

Report Page 5 

QUALITY ASSURANCE STATEMENT 

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

Harlan Laboratories Study: 

Test Item: 

Study Director: 

Study Title: 

C75560 

Leuco Sulfur Black 1 

Dr Martin KUhne 

Leuco Sulfur Black 1: Determination of the Water 

Solubility 

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 were periodically inspected. The study plan and this report were audited by the 

Quality Assurance. The dates are given below. 

Dates of Reports to the Study 

Dates and Types of QA Inspections Director and Test Facility 

Management 

February 10, 2010 Study plan February 10, 2010 

February 12, 2010 Process based February 12, 2010 (Work-up) 

March 09,2010 Report March 09,2010 

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

Quality Assurance: )~ Ms Margot Richter-Auer 
Harlan Laboratories Study C75560 

Leuco Sulfur Black l 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Reporting: 

Technical Coordinators: 

Head ofQA: 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Study Completion Date: 

Report 

Leuco Sulfur Black 1 

Leuco Sulfur Black 1: Determination of the Water 

Solubility 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Dr Bruno Kunz 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 Itingen I Switzerland 

Harlan Laboratories Ltd. 

Quality Assurance GLP 

Zelgliweg 1 

4452 Itingen I Switzerland 

Dr Martin Kiihne 

Dr Carsten Habeck 

Ms Christiane Nestler 

Ms Sabine Schlachter, Mr Johann Martens and Ms 

Barbara Fliikiger 

Mr Tobias Fink 

February 15, 2010 

February 19,2010 

March 11, 2010 

Page6 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

Report 

Data Requirements I Test Guidelines 

Page 7 

The study procedures indicated in this report meet or exceed the requirements of the following 

guidelines: 

• OECD Guideline for Testing of Chemicals, No. 105, "Water solubility"; adopted 

July 27, 1995. 

• EEC Directive 92/69, Part A, Methods for the determination of physico-chemical 

properties, A.6: "Water solubility", EEC Publication No. L383, December 1992. 

• Council Regulation (EC) No. 440/2008, 30 May 2008, Part A, Methods for the 

determination of physico-chemical properties, A.6 "Water Solubility" 

Archiving 

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

sample of test item(s), and the finalized report of the present study for at least ten years. 

No data will be discarded without the. Sponsor's written consent. 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

1 SUMMARY 

Report Page 8 

During this study the water solubility of Leuco Sulfur Black 1 was determined. Vessels 

containing water and test item were shaken for 24 hours, 48 hours and 72 hours, respectively, at 

30 °C. Afterwards the samples were equilibrated for another 24 hours at 20.0 °C. After dilution 

with water the concentrations in the aqueous solutions were determined by a TC-method. 

The water solubility of Leuco Sulfur Black 1 was determined to be 

62.4 mg/L at 20.0 °C and a pH of7.9 

based on a measured concentration of 

11.5 mg/L of carbon 

using the flask shaking method. 
Harlan Laboratories Study C75560 

Leuco Sulfur Black 1 

2 PURPOSE 

Report Page 9 

The purpose of this study was to evaluate the solubility of Leuco Sulfur Black 1 in water, which 

is specified by the saturation mass concentration of the test item in water at a given temperature. 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

Report Page 10 

3 MATERIALS AND METHODS 

3.1 Test Item 

Data of test item as supplied by the Sponsor. 

Identification: 

CASNr.: 

Description: 

Batch Number: 

Composition: 

Stability of the test item: 

Stability of the test item dilution: 

Expiry date: 

Storage Conditions: 

3.2 Test System 

Leuco Sulfur Black 1 

66241-11-0 

Thixotrophic, black, wet presscake 

ESA0007595 

Leuco Sulfur Black 1: 52.75% (calculated by amount of 

by-compounds) 

2,4-Dinitrochlorobenzene: < 20 ppm 

2,4-Dinitrophenole: < 20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water: 45.75% (w/w) by Karl-Fischer titration 

Stable under storage conditions 

> 4 h in water 

July 11, 20 11 

At room temperature (range 20 +!- 5 °C), light protected 

The test system consists of double distilled water. 

Rationale: The test systems and test methods are recommended by international guidelines. 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

3.3 Reagents and Apparatus 

1 ,4-Benzochinone 

Saccharose 

Water (used for dilution) 

Water (used for sample preparation) 

Filter Nylon (0.2 f.tm) 

Balances 

TC 

pH-meter 

Shaking Water Bath 

3.4 Principle 

Report 

Fluka 

Riedel de Haen 

Harlan Laboratories 

Harlan Laboratories 

BGB 

Mettler Toledo 

Shimadzu 

Metrohm 654 

Haake 

Infors AG 

Art. No. 12309 

Art. No. 16104 

Page II 

from Milli-Q water supply 

double distilled 

Art. No. F2503-1 

XP205DR 

Analyzer 5000A 

Metrohm electrode 6.0232.100 

SWB25 

Aquatron 

An excess amount of the test item is brought into contact with water. When saturation is 

achieved, the mass concentration of the test item in the aqueous solution is determined by a 

suitable analytical method. 

To determine the water solubility of the test item, two different methods can be used: 

- the column elution method 

- the flask method 

Due to the properties of the test item the flask method was used during this study. 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

3.5 Performance of the Test 

Test Solutions 

Report Page 12 

About 1.5 - 1. 7 g of test item were weighed into each of 6 Erlenmeyer flasks and 50 mL of 

double distilled water were added to each flask. The tightly closed flasks were shaken at about 

30 °C for 24 hours, 48 hours and 72 hours, respectively. Afterwards, the flasks were equilibrated 

for another 24 hours at 20.0 °C. The pH was measured to be about 7 .9. Afterwards, the 

supernatant solutions were centrifuged (~2900 g for 10 minutes), filtered (0.2 Jlm, Nylon) and 

5 mL of this solution was diluted up to 50 mL with water prior to analysis. The quantification 

was performed using TC measurement as described below. Details of sample treatment are given 

in Table 1. 

3.6 Analytical Method 

The concentration of the test item was analyzed using TC measurement. The test samples were 

quantified with reference to a calibration curve, which was generated automatically. The last 

calibration was performed on January 06, 2010. 

3.6.1 System Suitability check1 

A system suitability check was performed before sample measurement in order to check the 

reliability of the measurement. 

Preparation of 1,4-Benzochinone Solutions 

75.02 mg of 1,4-benzochinone were filled up to a volume of 1 L with Milli-Q water to get a 

stock solution of nominal concentration of 50 ppm of carbon. 2 mL of this stock solution was 

diluted to a volume of 200 mL with Milli-Q water to get a solution of a nominal concentration of 

0.5 ppm of carbon. A second solution of a nominal concentration of 10 ppm of carbon was 

prepared by diluting 20 mL of the stock solution up to 100 mL with Milli-Q water. For both 

solutions, the TOC was measured. The system suitability check was successfully as all TOC 

values were within the specifications. 

Preparation of Saccharose Solutions 

118.99 mg of saccharose were filled up to a volume of 1 L with Milli-Q water to get a stock 

solution of nominal concentration of 50 ppm of carbon. 2 mL of this stock solution was diluted 

to a volume of 200 mL with Milli-Q water to get a solution of a nominal concentration of 

0.5 ppm of carbon. Afterwards, the TOC was measured. The system suitability check was 

successfully as the TOC value was within the specifications. 

1 Was performed in Study C79744 
Harlan Laboratories Study C75560 

Leuco Sulfur Black 1 

3. 7 Data Handling 

Report Page 13 

The samples were quantified by Total Carbon (TC) measurement with reference to a calibration 

curve. The calibration curve was calculated using the software. The concentration of carbon of 

the injected samples was quantified by peak area with reference to the calibration curve. 

From the results obtained by the software the concentration of carbon in the analyzed solutions 

was calculated by the following equation: 

(1) 

Where 

C1 Measured concentration of carbon in the sample solution [ppm= mg/L] 

Cz Concentration of carbon in the test item solution before dilution [ mg/L] 

f Dilution factor 

From the concentration of carbon the water solubility of the test item was calculated by the 

following equation: 

(2) 

Where 

C3 Water solubility of the test item [mg/L] 

Cc Percentage of carbon in the test item as obtained by elementar analysis 

(here: 18.5%) 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

Report 

4 RESULTS AND DISCUSSION 

Page 14 

The determination of the water solubility of Leuco Sulfur Black 1 was performed according to 

the EEC Directive 92/69, A.6 "Water Solubility" (1992) and the OECD guideline No. 105, 

"Water Solubility" (1995) using the flask shaking method. 

After a shaking time of 24 hours, 48 hours and 72 hours, respectively, at 30 °C, the samples were 

equilibrated for another 24 hours at 20 °C. Afterwards, the samples were centrifuged, filtered, 

diluted and analyzed by TC measurement. The water solubility of Leuco Sulfur Black 1 at 20 °C 

was determined to be 62.4 mg/L. The measured results differ by less than 15%. Therefore, the 

study is considered to be valid. The results of the individual measurements of the main test are 

summarized in Table 2. The tabulated values represent rounded results, which were obtained by 

calculation using the exact raw data. 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

5 CONCLUSION 

Report Page 15 

During this study the water solubility of Leuco Sulfur Black 1 was determined and found to be 

62.4 mg/L at 20 °C and a pH of7.9 

based on a measured concentration of 

11.5 mg/L of carbon 

using the flask shaking method. 
Harlan Laboratories Study C75560 

Leuco Sulfur Black I 

6 TABLES 

Report 

Table 1 Data of the Performance of the Water Solubility Test 

Sample Solution No. 1 2 3 

Page 16 

4 5 6 

Weighed sample of the test 1482.52 1674.48 1473.65 1621.87 1734.69 1583.06 item [mg] 

Added volume of water [ mL] 50 50 50 50 50 50 

Agitation time at 30 oc 72 72 48 48 24 24 [hours] 

Equilibration time at 20 °C 24 24 24 24 24 24 [hours] 

pH value of test item solution 7.60 7.52 7.68 7.76 8.32 8.29 

Dilution factor before 10 10 10 10 10 10 quantification 

Table 2 Results of the Water Solubility ofLeuco Sulfur Black 1 

Con centra- Con centra- Con centra- Water 

Sample No. Shaking tion of tion of tion of test solubility 

time at 30 °C carbon, carbon, item, calculated 

analyzed calculated calculated (rounded) 

[mg!L] [mg/L] [mg/L] [mg/L] 

1 

72 

1.094 10.94 59.14 

2 1.400 14.00 75.68 

3 1.147 11.47 62.00 62.35 

48 SD = 6.90 4 1.104 11.04 59.68 

5 

24 

1.143 11.43 61.78 

6 1.033 10.33 55.84 

The saturation mass concentration is calculated from the bold values 

SD = standard deviation 

CONTA\NS NO CB\