### DISCLAIMER This report isthe propertyofClariant. Its content is confidential and notfor general release. It is not allowed to cite and reveal the report or its contentto a third partywithout priorwritten approval ofClariant . 

## REPORT 

## Leuco SulfurBlack 1 

# harlann·


# ....

Acute Toxicity ofLeuco SulfurBlack 1to Daphnia magna 

## in a 48-HourImmobilizationTest
StudyDirector: Test Facility:
Sponsor: Dr. A. Peither HarlanLaboratoriesLtd. Zelgliweg 1 4452 ltingen I 

### Switzerland ClariantProducts (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1 I 

### Switzerland StudyIdentification: Harlan Laboratories Study C31155 Version: Final StudyCompletion Date: 07-Aug-2009 Page 1of38 

### \ 

## 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 DataRequirements ITest Guidelines ....................................................................................8 Summary ofStudy 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 oftheImmobility, NOEC, ECo, ECso, and ECJOo............ l4 3.8.2 Water Quality Criteria............................................................................. 15 3.8.3 Analysis ofthe Test Item Concentrations ............................................... 15 4 RESULTS AND DISCUSSION.......................................................................................... 16 5 REFERENCES .................................................................................................................... 17 6 TABLES .............................................................................................................................. 18 APPENDIX I- FINALIZED STUDY PLAN AND FIRST AMENDMENTTO STUDY PLAN.....................................................................................................................20

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## LIST OFTABLES Table 1 Table 2 Table 3 Table 4 Table 5 Effect ofLeuco Sulfur Black 1onthe Mobility ofDaphnia magna.......................... 18 Appearance ofthe 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 3Harlan Laboratories Study C31155 Leuco SulfurBlack 1 

## GLP CERTIFICATE

Schweizensche Eidgenosstnschaft Confedfratlon suisse Confederazione Sviuera Confederaziun Pizra Report

Federal Department of Home Affairs DHA
,..

....Offlcaof PubUc HHith POPH federal Department of the Environment, Transpon, Energy and Communications OETEC**Peden!~** Offkefor the EnvlronnaentPO!N 

# / sw1ssrnedic 

## Statement ofGLP Compliance According to Article 14 paragraph 3Ordinance 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 compliancewith 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 ofthe testfacility: Areas ofexpertise according to article 3 paragraph 1JetterdOGLP: 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, 8.1Analytical and clinical chemistry testing, 9./Otherstudies (safety pharmacology and animal metabolism). Inspection authority: Federal Office forthe Environment (FOEN) I Federal Office of Public Health (FOPH) I SwissAgency for Therapeutic Products (Swissmedic) Date ofinspection: 05th to 09th and 26th to 30th November 2007 Date ofdecision: 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.authorityfor-chemicals

.**.\···\c~-3oo3~er'ri' ~** 

### 'Q~·('' ti{Jj_/JJjOJ 

## R&**,r~** * Bern, 12th November 08, The Head, Dr. Dag Kappes. n.no1ilk:etion aulhorltyforchltmlcU Ialhe OOCM"dnation R decision IUihartytor h good labcnloryptaclic. (GLP)far lhe FOEN, lhll FOPH MdSwlumedic. 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

Page4Harlan Laboratories Study C31155 Leuco SulfurBlack 1 GOOD LABORATORY PRACTICE Report



## STATEMENT OFCOMPLIANCE Harlan Laboratories Study: Test Item: StudyDirector: StudyTitle: C31155 Leuco Sulfur Black 1 Dr. A. 

### Peither Acute toxicity ofLeuco SulfurBlack 

### 1to Daphnia magna

### in a48-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 ofGood 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 affectedthe quality or integrity ofthe data. StudyDirector: Dr. A. Peither

Date:

## A.~J- 

# 9-( (oo9

Page 5Harlan Laboratories Study C3ll55 Leuco SulfurBlack I Report 

## QUALITYASSURANCE GLPSTATEMENT Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen I Switzerland Harlan Laboratories Study: Test Item: StudyDirector: C31155 Leuco SulfurBlack 1 Dr. A. Peither StudyTitle: Acute toxicity ofLeuco SulfurBlack 1to Daphnia magna in a 48-hourimmobilization test The general facilities and activities are inspected at least once a year and the results are reported to the responsible person andthe management. Studyprocedures were periodically inspected. The study plan andthis report were auditedbythe QualityAssurance. The dates are given below. Dates ofReports Dates and Types ofQA Inspections to theStudyDirector and TestFacility

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 reportreflects the raw data. Quality Assurance: Ms. S. Zeugin 

# _s. 

# 2p'.,.**o~t~:·**

# · 

# dj-;,11**·o~·**

# ··zoi>j.· · · ·

· · · · · · · · · · · · · ··

Page 6Harlan Laboratories Study C31155 Leuco SulfurBlack 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 1to 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 7Harlan Laboratories Study C31155 Leuco SulfurBlack 1 Report


### Data Requirements 

### ITest 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 of30 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 ofStudyPlan Amendments First Amendmentto Study Plan

Concerning: Alteration: Reason: Test Item Test item composition -Standardized test item information requested by the Sponsor -Reanalysis oftest item composition 

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

Page 8Harlan Laboratories Study C31155 Leuco SulfurBlack 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 ofimmobilized test organisms. The calculated concentration oftest 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 onthe test organisms is observed. Comprises test item treatments, control (test medium only) and solventcontrol (ifapplicable).

Page 9Harlan Laboratories Study C31155 Leuco SulfurBlack I 

## 1 SUMMARY Report

The acute toxicity ofthe test item Leuco SulfurBlack 1to 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 ofthe test item in test water. Due to the low water solubility ofthe test item, the test medium was prepared priorto the start of the test and prior to the test medium renewal as follows: a dispersion ofthe 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 ofDifficult Substances and Mixtures, 2000. No analytical work was performed due to the specific properties ofthe test item (insolubility in water). The biological results were based onthe loadingrate ofthetest item of 100 mg/L. In the control and at the loading rate of 1 00 mg/L no immobilized daphnids were determined duringthe test period of48 hours. The biological testresults (based onthe loading rate) were as follows: Loading rate (mg/L)

- 24-hour ECso: >100

- 24-hour ECo:**~100**

- 24-hourECwo: >100

- 48-hour ECso: >100

- 48-hourECo and

48-hourNOEC:**~100**

- 48-hourECwo: >100 In conclusion, the test item Leuco Sulfur Black 1had no acute toxic effects on Daphnia magna up to its solubility limit in the test water under the presenttest conditions. Page 10Harlan Laboratories Study C31155 Leuco SulfurBlack 1 

## 2 PURPOSE Report



### The purpose of the 48-hour toxicity test was to evaluate the effect of the test item Leuco SulfurBlack 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 leastthe loading rate of 100 mg!L.

Page 11Harlan Laboratories Study C3ll55 Leuco SulfurBlack l Report 

## 3 MATERIALSAND 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 SulfurBlack 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 ofthe 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 ofthe 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 ofthe test, the organisms used in the test were 6-24 hours old and were not first brood progeny. For evaluation ofthe quality ofthe daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result ofthe latestpositive control test in Februar 2009 (48-hour EC50: 0.71 mg/L, study C36915) indicated that the sensitivity ofthe 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 12Harlan Laboratories Study C31155 Leuco SulfurBlack I Report
The test method and the test species are recommended by the international test guidelines. 

### 3.3 TestWater 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 ofthe study until oxygen saturation was reached. Duringthe test period, the test water was not aerated. 

### 3.4 Material The test was performed in 100-mL glass beakers filled with 50 mL oftest medium. The test vessels were covered with glass plates to reduce the loss ofwaterby 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 watertemperature was maintained at 20 °C. A 16-hour lightto 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 l3Harlan Laboratories Study C31155 Leuco SulfurBlack I 

### 3.6 StudyDesign 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 ofthetest item of 100 mg/L was tested. Additionally, a control was tested in parallel (test water without addition ofthe test item). The limit test was based on the results ofa rangefinding test (non-GLP). A semi-static test with test medium renewal after 24 hours was performed to keep the concentration ofthe 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 ofthe corresponding concentration. 

### 3.7 Dosage Due to the low water solubility ofthe test item, dispersions with the loading rate of 100 mg/L were prepared before start ofthe test and before the renewal ofthe test medium by dispersing 100.3 and 100.0 mg of the test item in 1000 mL oftest water, respectively using ultrasonic treatment for 15 minutes and intense stirring. No auxiliary solvent oremulsifierwas used. The dispersions were stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum amount ofthe 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 ofdissolved test item were used as test medium at the start ofthe test(=addition ofdaphnids) and atthe testmedium renewal. The preparation ofthe test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures, 2000 [2]. 

### 3.8 Evaluations 3.8.1 Determination oftheImmobility,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 ofthe test beaker are consideredto be immobilized). The NOEC and ECo were determined directly from the raw data. The EC50 and the EC 100 could not be calculated due to the absence ofa toxic effect ofthe test item in this test. Page 14Harlan 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 watertemperature were determined in the test medium and in the control. The appearance ofthe test medium was visually recorded at the start ofthe test and after 24 and 48 hours. 

### 3.8.3 Analysis oftheTestItem Concentrations No analytical work was performed due to the specific properties ofthe test item (insolubility in water).

Page 15Harlan Laboratories Study C31155 Leuco SulfurBlack I Report 

## 4 RESULTSAND DISCUSSION A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect onDaphnia 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 testperiod of48 hours (Table I). Therefore, the 48-hour NOEC and the 48-hour EC0 ofLeuco Sulfur Black 1to Daphnia magna were determined to be the loading rate ofat least 100 mg/L. The 48-hourNOEC 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 EC 10owere clearly higherthan the loading rate of 100 mg/L. These values could not be quantified due to the absence of toxicity of Leuco Sulfur Black 1at the loading rateo f 100 mg/L. In conclusion, the test item Leuco Sulfur Black 1had no acute toxic effects on Daphnia magna up to its solubility limit in the test water under the presenttest conditions. No remarkable observations were made concerning the appearance ofthe test medium. It was clearthroughout the entire test duration (Table 2). At the beginning and end ofthe test medium renewal periods, the pH values ofthe 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 16Harlan Laboratories Study C31155 Leuco SulfurBlack 1 

# 5 REFERENCES 1. ISO Standard 6341 (1996): Report

Water quality- Determination ofthe inhibition ofthe mobility ofDaphnia magna Straus (Cladocera, Crustacea)- Acute toxicity test 2. OECD Series on Testing and AssessmentNo. 23 (2000): Guidance Document on Aquatic Toxicity Testing ofDifficultSubstances and Mixtures

Page 17Harlan Laboratories Study C31155 Leuco SulfurBlack I 

## 6 TABLES Report

Table 

### 1 Effect ofLeuco SulfurBlack 1on the Mobility ofDaphnia 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 ofthe TestMedium duringthe Test Period Abbreviations: 0: no remarkable observations, cleartest 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 ofthe test item 5: precipitation ofthetest item 6: test item at the surface 7: test item lying atthe bottom ofthe 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 18Harlan Laboratories Study C31155 Leuco SulfurBlack 1 Report
Table 3 pH Values in the Treatments Loadingrate Exposuretime 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 Exposuretime 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 Loadingrate Exposure time Oh 24 h 48 h (mg/L) new old new old Control 

### 20 20 20 20

100 20 20 20 20

Page 19Harlan Laboratories Study C31155 Leuco SulfurBlack I Report



## APPENDIX I- FINALIZED STUDYPLANAND FIRSTAMENDMENTTO STUDYPLAN

Page 20

# harlann· STUDY PLAN 

# ... Leuco Sulfur Black 1
Acute Toxicity ofLeuco Sulfur Black 1to Daphnia magna 

## in a 48-Hour ImmobilizationTest
StudyDirector: TestFacility:
Sponsor:

StudyIdentification: Version: Dr. A. Peither 

### HarlanLaboratoriesLtd. Zelgliweg 1 4452 ltingen I Switzerland 

### Clariant Products (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1 I Switzerland

Harlan Laboratories Study C31155 Final
Page 1of 13

Page 21Harlan Laboratories Study C31155 Leuco SulfurBlack 1 

## CONTENTS Study Plan Page 2

CONTENTS....................................................................................................................................2 SIGNATURES ................................................................................................................................3 PREFACE .......................................................................................................................................4 General Information...............................................................................................................4 Responsibilities......................................................................................................................4 Schedule.................................................................................................................................4 Good Laboratory Practice......................................................................................................5 DataRequirements ITest 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 ofthe ECO, EC50 and EC100.......................................... 11 2.8.2 Water Quality Criteria............................................................................. 11 2.8.3 Analysis ofthe Test Item Concentrations ............................................... 11 3 CONDITIONS FOR THE VALIDITY OF THE TEST...................................................... 12 4 REFERENCES .................................................................................................................... 12 5 REPORTING....................................................................................................................... 13 6 DISTRIBUTION ................................................................................................................. 13

Page 22Harlan Laboratories Study C31155 Leuco Sulfur Black 1 

## SIGNATURES StudyDirector:

Management:

Forthe Sponsor: Study Plan Page 3

Dr. A. Peither



# ..**~.ti~**

# ..................... Date:

## ~0.

# /)pnl'**2c?O~**
Dr. J. Magyar

Dr. U. Ensenbach ClariantProdukte (Deutschland) GmbH

# ...t(.~/d 

# .............................. Date:

Page 23Harlan 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 StartingDate: Anticipated Experimental Completion Date: Anticipated Date ofDraft Report: Study Plan

Leuco SulfurBlack 1 Acute toxicity ofLeuco SulfurBlack 

### 1to Daphnia magna in a 48-hour immobilizationtest 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 24Harlan Laboratories Study C31155 Leuco SulfurBlack 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 ofGood 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 

### ITest 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 ImmobilisationTest, 2004. • EU Commission Directive 92/69/EEC, C.2, Acute Toxicity for Daphnia, 1992. • Commission Regulation (EC) No 440/2008 of30 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 ofthe 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 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 Sponsorwill be promptly informed ofany relevant deviations from the study plan. 

### Archiving 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 withoutthe Sponsor's written consent.

Page 25Harlan Laboratories Study C31155 Leuco SulfurBlack 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 ofimmobilizedtest organisms. The calculated concentration oftest 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 effectonthe test organisms is observed. Comprises test item treatments, control (test medium only) and solvent control (ifapplicable).

Page 26Harlan 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 SulfurBlack 1 on the mobility of Daphnia magna. The EC50 at 48 hours will be calculated, ifapplicable. 

## 2 MATERIALSAND METHODS 2.1 Test Item The test item and the following information were provided by the Sponsor: Identity: CAS No.: BatchNo.: 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 SulfurBlack 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 orexcluded 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 sufficientto ensure personnel health and safety.

Page 27Harlan Laboratories Study C31155 Leuco SulfurBlack 1 

### 2.2 Test System Study Plan Page 8

The study will be performed with young daphnids ofthe species Daphnia magna Straus, which is the preferred test species recommended by the test guideline. A clone ofthis species was originally supplied by the University ofSheffield/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 ofthe 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 yearto demonstrate satisfactory conditions ofthe 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 waterto obtainthe 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:Mgand Na:K will be 4:1 and 10:1, respectively, based on molarity. Prior to the start ofthe 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 of7.6-8.0. The pH will normally not vary by more than 1.5 from the start until the end ofthe test.

Page 28Harlan 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 ofat least 50 mL will be used for the test. The test vessels will be covered with glass plates to reduce the loss ofwater by evaporation and to avoid the entry ofdust into the solutions. The test vessels will be labeled with the study number and all necessary additional informationto ensure unique identification. 

### 2.5 Experimental Conditions The test will be performed in a temperature-controlled room with continuous monitoring ofthe 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 StudyDesign A total of 20 daphnids per treatment will be used and divided into four replicates of five daphnids each. Providing a volume ofat 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 ofthe test. The duration ofthe test will be 48 hours. A semi-static test with a test medium renewal after 24 hours will be performed. A geometric series ofat least five concentrations differing by a constant factor not exceeding 2.2 will be used. The test concentrations should permit the determination ofthe 48-hour EC50 and, as far as possible, span the range ofO to 100% immobility (i.e., ECO and EC100). Concentrations ofthe test item higher than 100 mg/L or above the solubility limit ofthe 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 29Harlan Laboratories Study C31155 Leuco SulfurBlack 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 ofthe 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 ofCompliance in the test report. Pre-experiments may be performed within a different Harlan Laboratories study. However, the raw data or copies ofthe raw data ofthese tests will be archived under the study number ofthe present study. 

### 2.7 Dosage The test item is a complex mixture containing different sparingly soluble compounds. In orderto assess its toxicity, water accommodated fractions (WAFs) will be prepared. Individual dispersions ofthe test item will be prepared by direct addition ofrequisite 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 ofthe test and before each test medium renewal. Ifnecessary, pre-experiments will be performed to select suitable methods for the administration ofthe test item to test water and the preparation ofthe WAFs. The preparation ofthe test media will be based on the OECD Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures [2].

Page 30Harlan Laboratories Study C31155 Leuco SulfurBlack I 

### 2.8 Evaluations Study Plan

2.8.1 Determination ofthe ECO, ECSO and EClOO Page 11

The immobility of the daphnids will be determined by visual inspection after at least 24 and 48 hours ofexposure. Those organisms not able to swim within 15 seconds after gentle agitation ofthe testbeaker will be considered to be immobile. Ifapplicable, the 24-hour and 48-hour EC50 and their 95% confidence limits will be calculated e.g. by appropriate statistical tests as Probit Analysis [3, 4] or Moving Average Interpolation [5, 6]. The ECO, NOEC, and EClOO will be determined directly from the raw data, ifpossible. 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 ofthe Test Item Concentrations Onrequest ofthe sponsor, no analytical work will be performed.

Page 31Harlan Laboratories Study C31155 Leuco SulfurBlack 1 Study Plan 

## 3 CONDITIONS FORTHE VALIDITY OF THE TEST For the test to be valid, the following criteria should be fulfilled: Page 12

- In the control, not more than I0% ofthe daphnids should show immobilization or other signs ofdisease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).

- The dissolved oxygen concentration at the end ofthe test should be 2:3 mg/L in the control and test vessels. 

## 4 REFERENCES I. ISO Standard 6341 (1996): Water quality- Determination ofthe inhibition ofthe mobility ofDaphnia magna Straus (Cladocera, Crustacea)- Acute toxicity test 2. OECD Series on Testing and Assessment No. 23 (2000): Guidance Document on Aquatic Toxicity Testing ofDifficultSubstances and Mixtures 3. FINNEY, D.J. (1971): Probit Analysis, 3rd Edition, Cambridge University Press, London 4. DAVIES, R.G. (1971): ComputerProgramming in Quantitative Biology, Academic Press, London 5. THOMPSON, W.R., WElL, C.S. (1952): Onthe Construction ofTables for Moving Average Interpolation, Biometrics 8, 51-54 6. FINNEY, D.J. (1978): Statistical Methods in Biological Assay, 3rdEdition, Charles Griffin, London

Page 32Harlan 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 ofthe 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

# harianTh FIRSTAMENDMENTTO STUDYPLAN 

# ...
Leuco SulfurBlack 1
Acute Toxicity ofLeuco SulfurBlack 1toDaphnia magna 

## in a 48-Hour Immobilization Test
StudyDirector: TestFacility:
Sponsor:
StudyIdentification:
Version: Dr. A. Peither 

### HarlanLaboratoriesLtd. Zelgliweg 1 4452 ltingen I Switzerland 

### ClariantProducts (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1I Switzerland
Harlan Laboratories Study C31155
Final
Page 1of5

Page 34Harlan Laboratories Study C31155 Leuco Sulfur Black I 

### SIGNATURES StudyDirector:

Forthe Sponsor: FirstAmendmentto Study Plan Page2

Dr. A. Peither

Dr. U. Ensenbach ClariantProducts (Switzerland) Ltd.



# ...£...**~~**

# ... Date: 

# fv1' .? '(**I~~**1

Page 35Harlan Laboratories Study C3ll55 Leuco SulfurBlack I 

## PAGE 7 

## CONCERNING FirstAmendmentto 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: Expirationdate: Composition:

Purity: Stability: Storage conditions:
Safety precautions: Leuco SulfurBlack 1 66241-11-0 ESA0007595 Thixotropic, black, wet paste 11-July-2011

I. Leuco SulfurBlack 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 Atroom temperature (range 20 +/- 5°C), light protected. At Harlan Laboratories: atroom temperature, away from direct sunlight Routine hygienic procedures are sufficientto ensure personnel health and safety.

Page 36Harlan Laboratories Study C31155 Leuco SulfurBlack 1 

## NEW First Amendmentto Study Plan Page4

The test item and the following information concerning the test item were provided by the Sponsor: Identity: CAS No.: Description: BatchNo.: Composition:

Expiration date: Stability: Storage conditions: Leuco Sulfur Black 1 66241-11-0 Thixotropic, black, wetpaste 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-20II Stable under storage conditions At room temperature (range 20 

### ± 5°C), light protected. 

## REASON FORTHEALTERATION Standardized test item information requested by the Sponsor and additional test item information.

1Determined within Harlan Laboratories study C4344l Page 37Harlan Laboratories Study C31155 Leuco SulfurBlack I 

## DISTRIBUTION First Amendmentto Study Plan

This study plan amendment will be distributed as follows: Sponsor Sponsor: Harlan LaboratoriesLtd. Study File: Study Director: Deputy Study Director: Technical Coordinator: Logistics: 

### QAESM: 1 x copy
1 x (original) 

### 1x (copy) 1 x (copy) 1 x (copy) 1 x (copy) 1 x (copy) Page 5

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