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 

Leuco Sulfur Black 1 

harlann· .... 

Acute Toxicity ofLeuco Sulfur Black 1 to Daphnia magna in a 

48-Hour Immobilization Test 

Study Director: 

Test Facility: 

Sponsor: 

Dr. A. Peither 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Study Identification: Harlan Laboratories Study C31155 

Version: Final 

Study Completion Date: 07-Aug-2009 

Page 1 of38 

\ 

CONTAINS NO CBI 

( -\)~~ l-"J--
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

Report 

CONTENTS 

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

LIST OF TABLES .......................................................................................................................... 3 

GLP CERTIFICATE ....................................................................................................................... 4 

STATEMENT OF COMPLIANCE ................................................................................................ 5 

QUALITY ASSURANCE GLP STATEMENT ............................................................................. 6 

PREFACE ....................................................................................................................................... ? 

General Information ............................................................................................................... 7 

Responsibilities ...................................................................................................................... 7 

Schedule .................................................................................................................................. 7 

Data Requirements I Test Guidelines .................................................................................... 8 

Summary of Study Plan Amendments ................................................................................... 8 

Archiving ............................................................................................................................... 8 

DEFINITIONS ................................................................................................................................ 9 

I SUMMARY ......................................................................................................................... IO 

2 PURPOSE ............................................................................................................................ II 

3 MATERIALS AND METHODS ......................................................................................... l2 

3 .I Test Item ................................................................................................................ 12 

3.2 Test System ............................................................................................................ l2 

3.3 Test Water .............................................................................................................. l3 

3.4 Material .................................................................................................................. 13 

3.5 Experimental Conditions ....................................................................................... 13 

3.6 Study Design .......................................................................................................... 14 

3.7 Dosage ................................................................................................................... 14 

3.8 Evaluations ............................................................................................................ 14 

3.8.1 Determination ofthe Immobility, NOEC, ECo, ECso, and ECJOo ............ l4 

3. 8. 2 Water Quality Criteria ............................................................................. 15 

3.8.3 Analysis of the Test Item Concentrations ............................................... 15 

4 RESULTS AND DISCUSSION .......................................................................................... 16 

5 REFERENCES .................................................................................................................... 17 

6 TABLES .............................................................................................................................. 18 

APPENDIX I- FINALIZED STUDY PLAN AND FIRST AMENDMENT TO 

STUDY PLAN ..................................................................................................................... 20 

' "· •1 1 ~·· ''\"" 

~' ! '~.. ; 

· · "-'·Vi-V ',) <.' .. . rl1.. t ... .r~ .,. 

' ~;,:· 

Page 2 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

Report 

LIST OF TABLES 

Table 1 

Table 2 

Table 3 

Table 4 

Table 5 

Effect of Leuco Sulfur Black 1 on the Mobility of Daphnia magna .......................... 18 

Appearance of the Test Medium during the Test Period ............................................ 18 

pH Values in the Treatments ....................................................................................... 19 

Dissolved Oxygen Concentrations (mg/L) in the Treatments .................................... 19 

Temperature (°C) in the Treatments ........................................................................... 19 

Page 3 
Harlan Laboratories Study C3115 5 

Leuco Sulfur Black 1 

GLP CERTIFICATE 

Schweizensche Eidgenosstnschaft 

Confedfratlon suisse 

Confederazione Sviuera 

Confederaziun Pizra 

Report 

Federal Department of Home Affairs DHA 

,.. .... Offlca of PubUc HHith POPH 

federal Department of the Environment, 

Transpon, Energy and Communications OETEC 

Peden!~ Offke for the Envlronnaent PO!N 

/ sw1ssrnedic 

Statement of GLP Compliance 

According to Article 14 paragraph 3 Ordinance on Good Laboratory Practice [OGLP, SR 813.112.1) 

The notification authority for chemicals confirms that the following test facility was Inspected with 

respect to the compliance with the Swiss Ordinance on Good Laboratory Practice, adopted on 18th 

May 2005 [OGLP, SR 813.112.1). This Ordinance is based on the OECD Principles of Good 

Laboratory Practice, as revised in 1997 and adopted on 26th November 1997 by decision of the 

OECD Council [C(97)1861Final). 

Unequivocal name and address 

of the test facility: 

Areas of expertise according to 

article 3 paragraph 1 Jetter d OGLP: 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen, Switzerland. 

1./ Physical-chemical testing, 

2./ Toxicity studies, 

4./ Environmental toxicity studies on aquatic 

and terrestrial organisms, 

5./ Studies on behaviour In water, soli and air; 

bioaccumulation, 

6.1 Residue studies, 

7.1 Studies on effects on mesocosms and 

natural ecosystems, 

8.1 Analytical and clinical chemistry testing, 

9./ Other studies (safety pharmacology and 

animal metabolism). 

Inspection authority: Federal Office for the Environment (FOEN) I Federal Office of Public Health 

(FOPH) I Swiss Agency for Therapeutic Products (Swissmedic) 

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

Date of decision: 30th April 2008 

Based on the above mentioned decision It can be confirmed that the above mentioned test facility 

is able to conduct studies according to the aforemenUoned areas of expertise in compliance with 

the principles of GLP. The above mentioned test facility is listed in the register and GLP Jist 

according to the Article 14 OGLP and is Inspected on a regular basis according to Article 6 

paragraph 2 OGLP. 

Swiss Fedefl!l;c;Jff!ce of Public Health 

Consumer J:lrotection directorate 

Notification.authority for- chemicals 

. .\···\c~-3oo3~er'ri' ~ 

'Q~·('' ti{Jj_/JJjOJ R& ,r~ * 

Bern, 12th November 08, The Head, Dr. Dag Kappes. 

n. no1ilk:etion aulhorltyfor chltmlcU Ia lhe OOCM"dnation R decision IUiharty tor h good labcnlory ptaclic. (GLP) far lhe FOEN, lhll FOPH Md Swlumedic. 

SMu Fed'eraiO!flDeafPI.tlllcHeallh, C~~drwaorate, Noti!k:alkm &llhorityfot"ct'lenllollls, CH-30038em. 

www.g'P.~mnch Phone: +41 <Ol313227305 Fee +<11 l0l31 3236485 

Page4 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

GOOD LABORATORY PRACTICE 

Report 

STATEMENT OF COMPLIANCE 

Harlan Laboratories Study: 

Test Item: 

Study Director: 

Study Title: 

C31155 

Leuco Sulfur Black 1 

Dr. A. Peither 

Acute toxicity of Leuco Sulfur Black 1 to 

Daphnia magna in a 48-hour immobilization test 

This study has been performed in compliance witli 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]. 

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 (MHLW, MAFF and METI). 

Exclusions: 

Pre-experiments as mentioned in the report. 

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

Study Director: Dr. A. Peither 

Date: A.~J- 9-( (oo9 

Page 5 
Harlan Laboratories Study C3ll55 

Leuco Sulfur Black I 

Report 

QUALITY ASSURANCE GLP STATEMENT 

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

Harlan Laboratories Study: 

Test Item: 

Study Director: 

C31155 

Leuco Sulfur Black 1 

Dr. A. Peither 

Study Title: Acute toxicity of Leuco Sulfur Black 1 to 

Daphnia magna in a 48-hour immobilization test 

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 

Dates and Types of QA Inspections to the Study Director 

and Test Facility 

Management 

17-Apr-2009 Study Plan 17-Apr-2009 

31-Mar-2009 Process based (Application) 31-Mar-2009 

26-Jun-2009 Report 26-Jun-2009 

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

Quality Assurance: Ms. S. Zeugin 

_s. 2p'.,. o~t~:· · dj-;,11 ·o~· · ·zoi>j. · · · · · · · · · · · · · · · · · ·· 

Page 6 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Head ofQA: 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Report 

Leuco Sulfur Black 1 

Acute toxicity ofLeuco Sulfur Black 1 to 

Daphnia magna in a 48-hour immobilization test 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Dr. U. Ensenbach 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 Itingen I Switzerland 

Harlan Laboratories Ltd. 

Quality Assurance GLP 

Zelgliweg 1 

4452 ltingen I Switzerland 

Dr. A. Peither 

Dr. S. Hoger 

Mr. T. Fink 

08-May-2009 

14-May-2009 

Page 7 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

Report 

Data Requirements I Test Guidelines 

This study followed the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

• OECD Guideline for Testing of Chemicals, No. 202, Daphnia sp., Acute 

Immobilisation Test, 2004. 

• EU Commission Directive 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992. 

• Commission 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), C.2, Acute Toxicity for Daphnia. 

Summary of Study Plan Amendments 

First Amendment to Study Plan 

Concerning: Alteration: Reason: 

Test Item Test item composition -Standardized test item information requested by the 

Sponsor 

-Reanalysis of test item composition 

Archiving 

Harlan Laboratories Ltd. ( 4452 ltingen/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. 

Page 8 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

DEFINITIONS 

Immobilization: 

ECo: 

ECso: 

EC10o: 

NOEC (No Observed 

&_ffect ~oncentration): 

Treatment: 

Report 

Those organisms which are not able to swim within 

15 seconds after gentle agitation of the test container 

are considered to be immobile. 

The highest test concentration without a significant 

number of immobilized test organisms. 

The calculated concentration of test item which results 

in 50% immobilization. 

The lowest test concentration at which all test 

organisms are immobile. 

The highest test concentration at which no significant 

toxic effect on the test organisms is observed. 

Comprises test item treatments, control (test medium 

only) and solvent control (if applicable). 

Page 9 
Harlan Laboratories Study C3115 5 

Leuco Sulfur Black I 

1 SUMMARY 

Report 

The acute toxicity of the test item Leuco Sulfur Black 1 to Daphnia magna was determined in a 

48-hour semi-static test according to the EU Commission Directive 92/69/EEC, Part C.2, 

Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of 

Chemicals, No. 202 (2004). 

A limit test with a test medium renewal after 24 hours was performed in accordance with the test 

guidelines to demonstrate that the test item has no toxic effect on the test organisms up to the 

solubility limit of the test item in test water. 

Due to the low water solubility of the test item, the test medium was prepared prior to the start of 

the test and prior to the test medium renewal as follows: a dispersion of the test item with the 

loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room 

temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate of 

the dispersion was used as the test medium. Additionally, a control was tested in parallel. The 

preparation of the test medium was based on the OECD Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures, 2000. 

No analytical work was performed due to the specific properties of the test item (insolubility in 

water). 

The biological results were based on the loading rate ofthe test item of 100 mg/L. 

In the control and at the loading rate of 1 00 mg/L no immobilized daphnids were determined 

during the test period of 48 hours. 

The biological test results (based on the loading rate) were as follows: 

Loading rate 

(mg/L) 

- 24-hour ECso: >100 

- 24-hour ECo: ~100 

- 24-hour ECwo: >100 

- 48-hour ECso: >100 

- 48-hour ECo and 

48-hour NOEC: ~100 

- 48-hour ECwo: >100 

In conclusion, the test item Leuco Sulfur Black 1 had no acute toxic effects on Daphnia magna 

up to its solubility limit in the test water under the present test conditions. 

Page 10 
Harlan Laboratories Study C31155 

Leu co Sulfur Black 1 

2 PURPOSE 

Report 

The purpose of the 48-hour toxicity test was to evaluate the effect of the test item 

Leuco Sulfur Black 1 on the mobility of Daphnia magna. This limit test was performed in 

compliance with the test guidelines to demonstrate that the test item has no toxic effect on the 

test organisms up to at least the loading rate of 100 mg!L. 

Page 11 
Harlan Laboratories Study C3ll55 

Leuco Sulfur Black l 

Report 

3 MATERIALS AND METHODS 

3.1 Test Item 

The test item and the following information concerning the test item were provided by the 

Sponsor: 

Identity: 

CAS No.: 

Description: 

Batch No.: 

Composition: 

Expiration date: 

Stability: 

Storage conditions: 

3.2 Test System 

Leuco Sulfur Black 1 

66241-11-0 

Thixotropic, black, wet paste 

ESA0007595 

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

by-compounds) 1 

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) 1 

11-July-2011 

Stable under storage conditions 

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

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of 

this species (defined by the supplier as clone 5) was originally supplied by the University of 

Sheffield I UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in 

reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, 

main ions, and total hardness) and under temperature and light conditions identical to those of 

the tests (see below). 

At the start of the test, the organisms used in the test were 6-24 hours old and were not first 

brood progeny. 

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium 

dichromate is tested as a positive control twice a year. The result of the latest positive control test 

in Februar 2009 (48-hour EC50: 0.71 mg/L, study C36915) indicated that the sensitivity of the 

test organisms was within the internal historical range ( 48-hour ECso from 1996 to 2009: 

0.53-1.1 mg/L). 

1 Determined within Harlan Laboratories study C43441 

Page 12 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

Report 

The test method and the test species are recommended by the international test guidelines. 

3.3 Test Water 

Reconstituted test water according to ISO 6341 [ 1] was used in the study. It consisted of 

analytical grade salts dissolved in purified water at the following nominal concentrations: 

Ingredients I Concentration 

Properties mmol/L mg/L 

CaCh x 2H20 2.0 294 

MgS04 X 7H20 0.5 123 

NaHC03 0.75 65 

KCl 0.075 5.8 

Water Hardness 2.5 250 as CaC03 

Alkalinity 0.8 ---

The ratio ofCa:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. 

The test water was aerated prior to the start of the study until oxygen saturation was reached. 

During the test period, the test water was not aerated. 

3.4 Material 

The test was performed in 100-mL glass beakers filled with 50 mL of test medium. The test 

vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the 

entry of dust into the solutions. The test vessels were labeled with the study number and all 

necessary additional information to ensure unique identification. 

3.5 Experimental Conditions 

The test was performed in a temperature-controlled room with continuous monitoring of the 

room temperature. The water temperature was maintained at 20 °C. 

A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity 

during the light period was approximately between 520 and 680 Lux. 

The daphnids were not fed during the test. 

Page l3 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

3.6 Study Design 

Report 

A limit test was performed in accordance with the test guidelines to demonstrate that the test 

item has no toxic effect on the test organisms up to the loading rate of 100 mg/L. Thus, a single 

loading rate ofthe test item of 100 mg/L was tested. Additionally, a control was tested in parallel 

(test water without addition of the test item). The limit test was based on the results of a rangefinding test (non-GLP). 

A semi-static test with test medium renewal after 24 hours was performed to keep the 

concentration of the test item in the test medium as constant as possible during the test period of 

48 hours. After 24 hours, the test organisms were placed in clean test vessels with freshly 

prepared test medium of the corresponding concentration. 

3.7 Dosage 

Due to the low water solubility of the test item, dispersions with the loading rate of 100 mg/L 

were prepared before start of the test and before the renewal of the test medium by dispersing 

100.3 and 100.0 mg of the test item in 1000 mL of test water, respectively using ultrasonic 

treatment for 15 minutes and intense stirring. No auxiliary solvent or emulsifier was used. 

The dispersions were stirred by a magnetic stirrer at room temperature in the dark over 96 hours 

to dissolve a maximum amount of the test item in the respective dispersion. The long stirring 

period was chosen in consultation with the sponsor. 

After the 96-hour stirring period, the dispersions of the test item were filtered through a 

membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 ~m). The undiluted filtrates 

containing the maximum concentration of dissolved test item were used as test medium at the 

start of the test(= addition of daphnids) and at the test medium renewal. 

The preparation of the test medium was based on the OECD Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures, 2000 [2]. 

3.8 Evaluations 

3.8.1 Determination of the Immobility, NOEC, ECo, ECso, and EC10o 

The daphnids were observed for immobility after 24 and 48 hours of exposure ( daphnids not 

being able to swim within 15 seconds after gentle agitation of the test beaker are considered to be 

immobilized). 

The NOEC and ECo were determined directly from the raw data. 

The EC50 and the EC100 could not be calculated due to the absence of a toxic effect of the test 

item in this test. 

Page 14 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

3.8.2 Water Quality Criteria 

Report 

At the start and end of each test medium renewal period, the pH values, dissolved oxygen 

concentration and water temperature were determined in the test medium and in the control. 

The appearance of the test medium was visually recorded at the start of the test and after 24 and 

48 hours. 

3.8.3 Analysis of the Test Item Concentrations 

No analytical work was performed due to the specific properties of the test item (insolubility in 

water). 

Page 15 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

Report 

4 RESULTS AND DISCUSSION 

A limit test was performed in accordance with the test guidelines to demonstrate that the test 

item has no toxic effect on Daphnia magna at a loading rate of 100 mg/L. 

In the control and at the loading rate of 100 mg/L, no immobilized test organisms were observed 

during the test period of 48 hours (Table I). 

Therefore, the 48-hour NOEC and the 48-hour EC0 of Leuco Sulfur Black 1 to Daphnia magna 

were determined to be the loading rate of at least 100 mg/L. The 48-hour NOEC and the 48-hour 

EC0 might even be higher but loading rates above 100 mg/L were not tested, in accordance with 

the test guidelines. The 48-hour EC50 and the 48-hour EC10o were clearly higher than the loading 

rate of 100 mg/L. These values could not be quantified due to the absence of toxicity of 

Leuco Sulfur Black 1 at the loading rateo f 100 mg/L. 

In conclusion, the test item Leuco Sulfur Black 1 had no acute toxic effects on Daphnia magna 

up to its solubility limit in the test water under the present test conditions. 

No remarkable observations were made concerning the appearance of the test medium. It was 

clear throughout the entire test duration (Table 2). 

At the beginning and end of the test medium renewal periods, the pH values of the test medium 

were between 7.6 and 8.0 (Table 3). The dissolved oxygen concentrations in the test medium and 

control were at ·least 7.9 mg/L (Table 4), and water temperature was 20 oc during the test 

(Table 5). 

Page 16 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

5 REFERENCES 

1. ISO Standard 6341 (1996): 

Report 

Water quality- Determination of the inhibition of the mobility of Daphnia magna 

Straus (Cladocera, Crustacea)- Acute toxicity test 

2. OECD Series on Testing and Assessment No. 23 (2000): 

Guidance Document on Aquatic Toxicity Testing 

ofDifficult Substances and Mixtures 

Page 17 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

6 TABLES 

Report 

Table 1 Effect of Leuco Sulfur Black 1 on the Mobility of Daphnia magna 

No. of Immobilized Immobilized 

Loading rate daphnids daphnids after daphnids after 

tested 24 hours 48 hours 

(mg/L) No. % No. Ofo 

Control 20 0 0 0 0 

100 20 0 0 0 0 

Table 2 Appearance of the Test Medium during the Test Period 

Abbreviations: 

0: no remarkable observations, clear test medium 

1: homogeneous dispersion in the water, turbidity observable 

2: noticeable turbidity caused by the test item 

3: noticeable coloration caused by the test item 

4: inhomogeneous dispersion of the test item 

5: precipitation of the test item 

6: test item at the surface 

7: test item lying at the bottom of the test beaker 

Loading rate Exposure time 

Oh 24 h 48 h 

(mg/L) new old new old 

Control 0 0 0 0 

100 0 0 0 0 

Page 18 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

Report 

Table 3 pH Values in the Treatments 

Loading rate Exposure time 

Oh 24 h 

(mg!L) new old new 

Control 7.8 7.8 8.0 

100 7.6 7.6 8.0 

48 h 

old 

7.9 

8.0 

Table 4 Dissolved Oxygen Concentrations (mg/L) in the Treatments 

Loading rate Exposure time 

Oh 24 h 48 h 

(mg/L) new old new old 

Control 8.3 8.4 7.9 8.0 

100 8.0 8.1 8.0 8.1 

Table 5 Temperature eq in the Treatments 

Loading rate Exposure time 

Oh 24 h 48 h 

(mg/L) new old new old 

Control 20 20 20 20 

100 20 20 20 20 

Page 19 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

Report 

APPENDIX I- FINALIZED STUDY PLAN AND 

FIRST AMENDMENT TO STUDY PLAN 

Page 20 
harlann· 

STUDY PLAN ... 

Leuco Sulfur Black 1 

Acute Toxicity ofLeuco Sulfur Black 1 to Daphnia magna 

in a 48-Hour Immobilization Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

Version: 

Dr. A. Peither 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Harlan Laboratories Study C31155 

Final 

Page 1 of 13 

Page 21 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

CONTENTS 

Study Plan Page 2 

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

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

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

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

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

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

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

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

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

Archiving ............................................................................................................................... 5 

DEFINITIONS ................................................................................................................................ 6 

1 PURPOSE .............................................................................................................................. ? 

2 MATERIALS AND METHODS ........................................................................................... 7 

2.1 Test Item .................................................................................................................. 7 

2.2 Test System .............................................................................................................. 8 

2.3 Test Water ................................................................................................................ 8 

2.4 Material .................................................................................................................... 9 

2.5 Experimental Conditions ......................................................................................... 9 

2.6 Study Design ............................................................................................................ 9 

2.7 Dosage ................................................................................................................... 10 

2.8 Evaluations ............................................................................................................ 11 

2.8.1 Determination of the ECO, EC50 and EC100 .......................................... 11 

2.8.2 Water Quality Criteria ............................................................................. 11 

2.8.3 Analysis of the Test Item Concentrations ............................................... 11 

3 CONDITIONS FOR THE VALIDITY OF THE TEST ...................................................... 12 

4 REFERENCES .................................................................................................................... 12 

5 REPORTING ....................................................................................................................... 13 

6 DISTRIBUTION ................................................................................................................. 13 

Page 22 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

SIGNATURES 

Study Director: 

Management: 

For the Sponsor: 

Study Plan Page 3 

Dr. A. Peither 

.. ~.ti~ .................... . 

Date: ~0. /)pnl' 2c?O~ 

Dr. J. Magyar 

Dr. U. Ensenbach 

Clariant Produkte (Deutschland) GmbH 

...t(.~/d ............................. . Date: 

Page 23 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Head ofQA: 

Schedule 

Anticipated Experimental 

Starting Date: 

Anticipated Experimental 

Completion Date: 

Anticipated Date of Draft Report: 

Study Plan 

Leuco Sulfur Black 1 

Acute toxicity of Leuco Sulfur Black 1 to 

Daphnia magna 

in a 48-hour immobilization test 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz I I Switzerland 

Dr. U. Ensenbach 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen I Switzerland 

Harlan Laboratories Ltd. 

Quality Assurance GLP 

Zelgliweg 1 

4452 Itingen I Switzerland 

Dr. A. Peither 

Dr. S. Hoger 

Mr. T. Fink 

Apr-2009 

Apr/May-2009 

May-2009 

Page4 

Page 24 
Harlan Laboratories Study C3115 5 

Leuco Sulfur Black 1 

Good Laboratory Practice 

Study Plan Page 5 

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

Laboratory Practice, adopted May 181

\ 2005 [SR 813.112.1]. This Ordinance is based on the 

OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 261

h, 

1997 by decision ofthe OECD Council [C(97)186/Fina1]. 

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 (MHLW, MAFF and METI). 

Data Requirements I Test Guidelines 

The study will be performed according to the following internationally accepted guidelines and 

recommendations: 

• OECD Guideline for Testing of Chemicals, No. 202, Daphnia sp., Acute 

Immobilisation Test, 2004. 

• EU Commission Directive 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992. 

• Commission 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), C.2, Acute Toxicity for Daphnia. 

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 the Study Director's 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. 

Archiving 

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. 

Page 25 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

DEFINITIONS 

Immobilization: 

ECO: 

EC50: 

EClOO: 

NOEC (No Observed 

~ffect h.oncentration): 

Treatment: 

Study Plan Page 6 

Those organisms which are not able to swim within 

15 seconds after gentle agitation of the test container 

are considered to be immobile. 

The highest test concentration without a significant 

number of immobilized test organisms. 

The calculated concentration of test item which results 

in 50% immobilization. 

The lowest test concentration at which all test 

organisms are immobile. 

The highest test concentration at which no significant 

toxic effect on the test organisms is observed. 

Comprises test item treatments, control (test medium 

only) and solvent control (if applicable). 

Page 26 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

1 PURPOSE 

Study Plan Page 7 

The purpose of the 48-hour toxtctty test is to evaluate the effect of the test item 

Leuco Sulfur Black 1 on the mobility of Daphnia magna. The EC50 at 48 hours will be 

calculated, if applicable. 

2 MATERIALS AND METHODS 

2.1 Test Item 

The test item and the following information were provided by the Sponsor: 

Identity: 

CAS No.: 

Batch No.: 

Description: 

Expiration date: 

Composition: 

Purity: 

Stability: 

Storage conditions: 

Safety precautions: 

Leuco Sulfur Black 1 

66241-11-0 

ESA0007595 

Thixotropic, black, wet paste 

11-July-20 11 

I. Leuco Sulfur Black I: will be amended 

II. 2,4-Dinitrochlorobenzene: <20 ppm 

III. 2,4-Dinitrophenole: <20 ppm 

IV. Sulfate: 970 ppm 

V. Chloride: 750 ppm 

VI. Sodium: 1.3 % (w/w) 

VII. Water: will be amended 

Will be amended or excluded from the Statement of 

Compliance. 

Stable under storage conditions 

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

protected. 

At Harlan Laboratories: at room temperature, away 

from direct sunlight 

Routine hygienic procedures are sufficient to ensure 

personnel health and safety. 

Page 27 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

2.2 Test System 

Study Plan Page 8 

The study will be performed with young daphnids of the species Daphnia magna Straus, which 

is the preferred test species recommended by the test guideline. A clone of this species was 

originally supplied by the University of Sheffield/UK in 1992. The clone (defined by the supplier 

as clone 5) is successfully bred in Harlan Laboratories under temperature and light conditions 

identical to those of the tests. The cultivation of the parental daphnids is performed in 

reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, 

main ions, and total hardness). 

At the start of the test, the test organisms will be :::;24 hours old and will not be first brood 

progeny. 

For evaluation of the quality of the Daphnia magna clone and the experimental conditions, 

potassium dichromate will be tested as a positive control twice a year to demonstrate satisfactory 

conditions of the test. 

The test method and the test species are recommended by the international test guidelines. 

2.3 Test Water 

Reconstituted test water according to ISO 6341 [ 1] will be used in the study. It will consist of 

analytical grade salts dissolved in purified water to obtain the following nominal concentrations: 

Ingredients I Concentration 

Properties mmoi/L mg/L 

CaCh x 2H20 2.0 294 

MgS04 X 7H20 0.5 123 

NaHC03 0.75 65 

KCl 0.075 5.8 

Water Hardness 2.5 250 as CaC03 

Alkalinity 0.8 ---

The ratio ofCa:Mg and Na:K will be 4:1 and 10:1, respectively, based on molarity. 

Prior to the start of the study, the test water will be aerated until oxygen saturation is reached. 

Aeration will not be performed during the experiment. The pH of the test water after 

equilibration will be in the range of 7 .6-8.0. The pH will normally not vary by more than 1.5 

from the start until the end of the test. 

Page 28 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

2.4 Material 

Study Plan Page 9 

Glass test vessels (e.g. beakers or test tubes) with a volume of at least 50 mL will be used for the 

test. The test vessels will be covered with glass plates to reduce the loss of water by evaporation 

and to avoid the entry of dust into the solutions. 

The test vessels will be labeled with the study number and all necessary additional information to 

ensure unique identification. 

2.5 Experimental Conditions 

The test will be performed in a temperature-controlled room with continuous monitoring of the 

room temperature. The water temperature will be maintained at 18-22 oc with a maximum 

deviation of± 1 oc during the test. 

A 16-hour light to 8-hour dark cycle will be used with a 30-minute transition period. Light 

intensity during the light period will be between 200 and 1200 Lux and will be documented in 

the report. 

The daphnids will not be fed during the test. 

2.6 Study Design 

A total of 20 daphnids per treatment will be used and divided into four replicates of five 

daphnids each. Providing a volume of at least 50 mL test medium per replicate, at least 2 mL of 

test medium will be provided for each daphnia as required by the test guidelines. The daphnids 

will be randomly distributed to the test vessels at initiation of the test. The duration of the test 

will be 48 hours. 

A semi-static test with a test medium renewal after 24 hours will be performed. 

A geometric series of at least five concentrations differing by a constant factor not exceeding 2.2 

will be used. The test concentrations should permit the determination of the 48-hour EC50 and, 

as far as possible, span the range ofO to 100% immobility (i.e., ECO and EC100). Concentrations 

of the test item higher than 100 mg/L or above the solubility limit of the test item in the test 

water (whichever is the lower) will usually not be tested in accordance with the test guidelines. 

A control (test water without test item) will be run in parallel to the test concentrations. The 

control may also be used for other studies running in parallel at Harlan Laboratories. 

Page 29 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

Study Plan Page 10 

Normally, a range-finding test (non-GLP) will be conducted in order to determine the range of 

concentrations to be used. If no significant toxic response is noted at the highest test 

concentration or loading rate of 100 mg/L or at the highest concentration of the test item that can 

be applied, only this concentration and the corresponding control will be tested in the definitive 

test (Limit Test according to the test guidelines). 

In general, the range-finding test and any pre-experiment are not performed in compliance with 

the GLP Regulations and will therefore be excluded from the Statement of Compliance in the 

test report. Pre-experiments may be performed within a different Harlan Laboratories study. 

However, the raw data or copies of the raw data of these tests will be archived under the study 

number of the present study. 

2.7 Dosage 

The test item is a complex mixture containing different sparingly soluble compounds. In order to 

assess its toxicity, water accommodated fractions (WAFs) will be prepared. 

Individual dispersions of the test item will be prepared by direct addition of requisite amounts of 

test item to test water. No auxiliary solvent or emulsifier will be used. The test item will be 

mixed into the test water as homogeneously as possible using, for example, ultrasonic treatment 

and/or intense stirring. 

The dispersions will be stirred on magnetic stirrers at room temperature over 96 hours to dissolve 

maximum amounts of the different compounds of the test item in the dispersions. The long 

stirring time of the dispersion was chosen since the test item is stable in water (information 

provided by the sponsor). 

After stirring, dispersions will be filtered through membrane filters (Schleicher & Schuell, Type 

NC45, pore size 0.45 J..Lm) just before the start of the test. The filtrates of the equilibrated 

dispersions will be tested as WAFs. 

The test media will be freshly prepared before the start of the test and before each test medium 

renewal. 

If necessary, pre-experiments will be performed to select suitable methods for the administration 

of the test item to test water and the preparation of the WAFs. 

The preparation of the test media will be based on the OECD Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures [2]. 

Page 30 
Harlan Laboratories Study C31155 

Leuco Sulfur Black I 

2.8 Evaluations 

Study Plan 

2.8.1 Determination of the ECO, ECSO and EClOO 

Page 11 

The immobility of the daphnids will be determined by visual inspection after at least 24 and 

48 hours of exposure. Those organisms not able to swim within 15 seconds after gentle agitation 

of the test beaker will be considered to be immobile. 

If applicable, the 24-hour and 48-hour EC50 and their 95% confidence limits will be calculated 

e.g. by appropriate statistical tests as Pro bit Analysis [3, 4] or Moving Average Interpolation 

[5, 6]. The ECO, NOEC, and EClOO will be determined directly from the raw data, if possible. 

2.8.2 Water Quality Criteria 

The pH values, dissolved oxygen concentrations and water temperatures will be measured in all 

treatments at least at the start and end of the test. The appearance of the test media will be 

recorded at least daily. 

2.8.3 Analysis of the Test Item Concentrations 

On request of the sponsor, no analytical work will be performed. 

Page 31 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

Study Plan 

3 CONDITIONS FOR THE VALIDITY OF THE TEST 

For the test to be valid, the following criteria should be fulfilled: 

Page 12 

- In the control, not more than I 0% of the daphnids should show immobilization or other 

signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the 

surface water). 

- The dissolved oxygen concentration at the end of the test should be 2:3 mg/L in the 

control and test vessels. 

4 REFERENCES 

I. ISO Standard 6341 ( 1996): 

Water quality- Determination of the inhibition of the mobility of Daphnia magna 

Straus (Cladocera, Crustacea)- Acute toxicity test 

2. OECD Series on Testing and Assessment No. 23 (2000): 

Guidance Document on Aquatic Toxicity Testing 

ofDifficult Substances and Mixtures 

3. FINNEY, D.J. (1971): 

Probit Analysis, 

3rd Edition, Cambridge University Press, London 

4. DAVIES, R.G. (1971): 

Computer Programming in Quantitative Biology, 

Academic Press, London 

5. THOMPSON, W.R., WElL, C.S. (1952): 

On the Construction ofTables for Moving Average Interpolation, 

Biometrics 8, 51-54 

6. FINNEY, D.J. (1978): 

Statistical Methods in Biological Assay, 

3rd Edition, Charles Griffin, London 

Page 32 
Harlan Laboratories Study C3ll55 

Leuco Sulfur Black l 

5 REPORTING 

Study Plan Page l3 

A GLP-compliant draft report will be submitted to the Sponsor for scientific review. Following 

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

6 DISTRIBUTION 

This study plan will be distributed as follows: 

Sponsor 

Monitor I Sponsor's representative: 

Harlan Laboratories Ltd. 

Study File: 

Study Director: 

Technical Coordinator: 

Logistics: 

QAESM: 

1 x copy 

1 x original 

1 x copy 

1 x copy 

1 x copy 

1 x copy 

Page 33 
haria nTh 

FIRST AMENDMENT TO STUDY PLAN ... 

Leuco Sulfur Black 1 

Acute Toxicity ofLeuco Sulfur Black 1 to Daphnia magna in a 

48-Hour Immobilization Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

Version: 

Dr. A. Peither 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Harlan Laboratories Study C31155 

Final 

Page 1 of5 

Page 34 
Harlan Laboratories Study C311 55 

Leuco Sulfur Black I 

SIGNATURES 

Study Director: 

For the Sponsor: 

First Amendment to Study Plan Page2 

Dr. A. Peither 

Dr. U. Ensenbach 

Clariant Products (Switzerland) Ltd . 

... £. .. ~~ ... Date: fv1' .? '( I~~ 1 

Page 35 
Harlan Laboratories Study C3ll55 

Leuco Sulfur Black I 

PAGE 

7 

CONCERNING 

First Amendment to Study Plan Page 3 

2 MATERIALS AND METHODS 

2.1 Test Item 

PRESENT 

The test item and the following information were provided by the Sponsor: 

Identity: 

CAS No.: 

Batch No.: 

Description: 

Expiration date: 

Composition: 

Purity: 

Stability: 

Storage conditions: 

Safety precautions: 

Leuco Sulfur Black 1 

66241-11-0 

ESA0007595 

Thixotropic, black, wet paste 

11-July-2011 

I. Leuco Sulfur Black I: will be amended 

II. 2,4-Dinitrochlorobenzene: <20 ppm 

III. 2,4-Dinitrophenole: <20 ppm 

IV. Sulfate: 970 ppm 

V. Chloride: 750 ppm 

VI. Sodium: 1.3 % (w/w) 

VII. Water: will be amended 

Will be amended or excluded from the Statement of 

Compliance. 

Stable under storage conditions 

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

protected. 

At Harlan Laboratories: at room temperature, away 

from direct sunlight 

Routine hygienic procedures are sufficient to ensure 

personnel health and safety. 

Page 36 
Harlan Laboratories Study C31155 

Leuco Sulfur Black 1 

NEW 

First Amendment to Study Plan Page4 

The test item and the following information concerning the test item were provided by the 

Sponsor: 

Identity: 

CAS No.: 

Description: 

Batch No.: 

Composition: 

Expiration date: 

Stability: 

Storage conditions: 

Leuco Sulfur Black 1 

66241-11-0 

Thixotropic, black, wet paste 

ESA0007595 

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

by-compounds) 1 

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) 1 

11-July-20 II 

Stable under storage conditions 

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

REASON FOR THE ALTERATION 

Standardized test item information requested by the Sponsor and additional test item information. 

1 Determined within Harlan Laboratories study C4344l 

Page 37 
Harlan Laboratories Study C3115 5 

Leuco Sulfur Black I 

DISTRIBUTION 

First Amendment to Study Plan 

This study plan amendment will be distributed as follows: 

Sponsor 

Sponsor: 

Harlan Laboratories Ltd. 

Study File: 

Study Director: 

Deputy Study Director: 

Technical Coordinator: 

Logistics: 

QAESM: 

1 x copy 

1 x (original) 

1 x (copy) 

1 x (copy) 

1 x (copy) 

1 x (copy) 

1 x (copy) 

Page 5 

Page 38