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

## REPORT Leuco Sulfur Black 1
Acute Toxicity ofLeuco SulfurBlack 1to Zebra Fish 

## (Brachydanio rerio) in a 96-HourTest
StudyDirector: TestFacility:
Sponsor: Dr. A. Peither HarlanLaboratories Ltd. Zelgliweg 1 4452 Itingen 

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

### I Switzerland StudyIdentification: Harlan Laboratories Study C31144 Version: Final StudyCompletion Date: 07-Aug-2009 Page 1 of40 

## CONTAINS NO CBt

# ('-PH~ 

# "r:;_1; l--:2Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## CONTENTS Report

CONTENTS....................................................................................................................................2 LIST OF TABLES ..........................................................................................................................3 GLP CERTIFICATE.......................................................................................................................4 STATEMENT OF COMPLIANCE................................................................................................5 QUALITY ASSURANCEGLP STATEMENT.............................................................................6 PREFACE .......................................................................................................................................? General Information...............................................................................................................7 Responsibilities......................................................................................................................7 Schedule.................................................................................................................................7 DataRequirements 

### ITest Guidelines ....................................................................................8 Animal Welfare......................................................................................................................8 Summary ofStudyPlan Amendments...................................................................................8 Archiving ...............................................................................................................................8 DEFINITIONS ................................................................................................................................9 1 SUMMARY......................................................................................................................... 10 2 PURPOSE............................................................................................................................ 11 3 MATERIALS AND METHODS......................................................................................... 12 3.1 Test Item ................................................................................................................ 12 3.2 Test System............................................................................................................ 12 3.3 TestWater.............................................................................................................. 13 3.4 Material.................................................................................................................. 13 3.5 Experimental Conditions ....................................................................................... 13 3.6 StudyDesign.......................................................................................................... 14 3.7 Dosage ................................................................................................................... 14 3.8 Evaluations ............................................................................................................ 15 3.8.1 Determination ofthe LC50, LCO, LClOO, NOEC and LOEC ................ 15 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 AMENDMENT TO STUDY PLAN.....................................................................................................................21

Page2Harlan Laboratories Study C31144 Leuco SulfurBlack I Report 

## LIST OFTABLES Table 1: Mortality and Visible Abnormalities Observed atthe TestFish ................................ 18 Table 2: Appearance ofthe Freshly Prepared and OldTestMedium....................................... 19 Table 3: pHinthe FreshlyPrepared and Old TestMedium and in the Control .......................20 Table4: Oxygen Concentrations (mg/L} in the Freshly Prepared and Old Test Medium and in the Control.........................................................................................20 Table 5: Temperatures 

## ec) inthe FreshlyPrepared and OldTestMedium and in the Control ..............................................................................................................20

Page 3Harlan Laboratories Study C31144 Leuco SulfurBlack l 

## GLPCERTIFICATE

11'11 Schwelzerlsche lldgenossenschift y Conf!ch!ration suisse Confederazlone Svh:zera Conftderazlun svixra Report

Federal Department of Homt Affairs DHA Pederal Offlce ofPublic Hnhh POPH ftdtrallnpartmtnt of the Environment. Transport. Energy and Communlcaclons DETEC**Peden~**Oftkefortht !nvlrornent PO!II 

# /' SWlSSmedk 

## 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 respectto 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 Pracllce, as revised In 1997 and adopted on 26th November 1997 by decision ofthe OECD Council [C(97)1861Final]. Unequivocal name and address ofthe testfacility: Harlan Laboratories Ltd. Zelgliweg 1 4452 ltingen, Switzelland. Areas ofexpertise according to article 3 paragraph 1Jetter dOGLP: 1JPhysical-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./ Residue studies, 7.IStudies 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 forthe Environment (FOEN) I Federal Office of Public HeaHh (FOPH) ISwiss Agency forTherapeutic Products (Swissmedlc) Data ofInspection: 05th to 09th and 26th to 3oth November2007 Date ofdecision: 30th April 2008 Based on the above mentioned decision Hcan be confirmed that the above mentioned test facility is able to conduct studies according to the aforementioned areas of expertise in compliance with the principles of GLP. The above mentioned test facUlty Is listed In the register and GLP list according to the Article 14 OGLP and Is Inspected on a regular basis according to Article 6 paragraph 2 OGLP.

1htnoc11c1t1Dn aultlodlyfotehllnlelfS IaINIc:oan:lnllllonand decWon auttol)'far ..goodlabonllorypracllct (Gl.P)far h FOEN, ..FOPH llldSwlamedlc. 8MnFldatal Qmcaat**P~Htlllh.** Conunerprotectlonclnlct:cnle,**H~aullodtyfarc:hlalll*s,** CH.aoD3lem. w.mplp·!dm"'!!h: PhDn« <t41 ((!)313227305 Fax: +41 f013132354118

Page4Harlan Laboratories Study C31144 Leuco SulfurBlack 1 GOOD LABORATORY PRACTICE Report



## STATEMENT OF COMPLIANCE Harlan Laboratories Study: Test Item: StudyDirector: StudyTitle: C31144 Leuco Sulfur Black 1 Dr. A. Peither Acute toxicity ofLeuco SulfurBlack 1to zebra fish (Brachydanio rerio) 

### in a 96-hourtest 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 261 \ 

### 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 (MHL W, MAFF and MET!). Exclusions:

- Pre-experiments as mentioned in the report. There were no circumstances that may have affected the quality orintegrity ofthe data. Study Director: Dr. A. 

### Peither



# ..i ..**t.~~**

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

# fot**3 ~**  

# 1, 2.()() <(

Page 5Harlan Laboratories Study C31144 Leuco Sulfur Black I Report 

## QUALITYASSURANCE GLPSTATEMENT Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen 

### I Switzerland Harlan Laboratories Study: Test Item: Study Director: C31144 Leuco Sulfur Black 1 Dr. A. Peither Study Title: Acute toxicity ofLeuco SulfurBlack 1to zebra fish (Brachydanio rerio) in a 96-hourtest The general facilities and activities are inspected at least once a year and the results are reported to theresponsible person andthe management. Study procedures were periodically inspected. The study plan and this report were auditedbythe Quality Assurance. The dates are given below. 

### Dates ofReports Dates andTypes ofQA Inspections to the Study

### Director and to the TestFacility Management 20-Apr-2009 Studyplan 20-Apr-2009 20-Apr-2009 Process based (test conditions) 20-Apr-2009 26-Jun-2009 Report 26-Jun-2009 This statement also confirms thatthis final reportreflects the raw data. Quality Assurance: Ms. S. Zeugin 

## ___s..

# 27'""

·····························································

Page 6Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## PREFACE General Information TestItem: StudyTitle: Sponsor: StudyMonitor: TestFacility: QA:


### Responsibilities StudyDirector: Deputy Study Director: HeadofQA: 

### Schedule Experimental Starting Date: Experimental Completion Date: Report

Leuco Sulfur Black 1 Acute toxicity ofLeuco Sulfur Black 1to zebra fish (Brachydanio rerio) in a 96-hourtest 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 Itingen I Switzerland

Dr. A. Peither Dr. S. Hoger Mr. 

### T. Fink
04-May-2009 15-May-2009

Page?Harlan Laboratories Study C31144 Leuco SulfurBlack 1 Report


### Data Requirements 

### I Test Guidelines This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: • OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 1992. • EU Commission Directive 92/69/EEC, C.l: Acute Toxicity for Fish, 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.1: Acute Toxicity for Fish. 

### Animal Welfare The study was performed in an AAALAC-accredited laboratory in accordance with the Swiss Animal Protection Law under license No. 59 (range finding test) and license No. 60 (definitive test). 

### Summary ofStudyPlan Amendments First Amendment to StudyPlan

Concerning: Alteration: Reason: Test Item Test item composition -Standardized test item informationrequested by the Sponsor -Reanalysis oftestitem 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 least ten years. No data will be discarded withoutthe Sponsor's written consent.

Page 8Harlan Laboratories StudyC31144 Leuco SulfurBlack 1 

## DEFINITIONS LCO: LC50:
LClOO: NOEC (No Observed Effect !;.oncentration): LOEC (Lowest Observed Effect !;.oncentration): Treatment: Report



### The highest test concentration without a significant number ofdead test organisms. The median lethal concentration, i.e. the calculated concentration oftest item which causes death in 50% of the test organisms. The lowest test concentration at which all test organisms are dead. The highest test concentration at which no significant toxic effect on the test organisms is observed. The lowest test concentration at which a significant toxic effect on the test organisms is observed. Comprises test item treatments, control (test medium only) and solvent control (ifapplicable).

Page 9Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## 1 SUMMARY Report

The acute toxicity ofthe test item Leuco Sulfur Black I to zebra fish (Brachydanio rerio) was determined in a 96-hour semi-static test with a daily test medium renewal according to the Commission Regulation (EC) No 440/2008, Part C.l and the OECD Guideline for Testing of ChemicalsNo. 203 (1992). A limit test with a daily test medium renewal was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L. Due to the low water solubility ofthe test item, the test medium was preparedpriorto the start of the test and prior to the test medium renewals as follows: a dispersion ofthe test item with the loading rate of 100 mg/L was continuously stirred atroom temperature in the darkover 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the maximum concentration of dissolved test item was used as the test medium. Additionally, a control was tested in parallel. The preparation ofthe test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing ofDifficult Substances andMixtures, 2000 [1]. No analytical work was performed due to the specific properties ofthe test item (insolubility in water). Thebiological results werebased on the loading rate ofthe test item. In the control and at the loading rate ofthe test item of 100 mg/L no mortality or other visible abnormalities were determined during the testperiod of96 hours. The summary ofthebiological results is as follows:

- 96-hourLC50:

- 96-hourLCO:

- 96-hourLClOO:

- 96-hourNOEC:

- 96-hour LOEC: Loading rate (mg/L)

>100**~100** >100**~100** >100 In conclusion, the test item had no acute toxic effects on the test fish up to the loading rate of 100 mg/L.

Page 10Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## 2 PURPOSE Report



### The purpose of this 96-hour toxicity test was to evaluate the acute toxicity of the test item Leuco SulfurBlack 1to fish. For this purpose, zebra fish (Brachydanio rerio) 

### were exposed in a semi-static test to an aqueous test medium at a loading rate of 100 mg/L under defined conditions. This limit test was based on a range-finding test and was performed in compliance with the test guidelines to demonstrate that the test item has no toxic effect on fish up to at least this concentration. The test fish were observed for mortality and visible abnormalities.

Page 11Harlan Laboratories Study C31144 Leuco SulfurBlack I 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:

Stability ofTest Item: ExpiryDate: Storage Conditions: 

### 3.2 Test System Leuco SulfurBlack 1 66241-11-0 Thixotrophic, black, wetpaste ESA0007595 Leuco SulfurBlack 1: 52.75% (calculatedbyamount of by-compounds) 2,4-Dinitrochlorobenzene: <20 ppm 2,4-Dinitrophenole: <20 ppm Sulfate: 970 ppm Chloride: 750 ppm Sodium: 1.3% (w/w) Water: 45.75% (w/w) Stable under storage conditions 11-Jul-2011 At room temperature (range 20 ± 5°C}, lightprotected.

The study was performed with zebra fish (Brachydanio rerio). The fish were obtained from a breeding culture at Harlan Laboratories. No medication was applied during holding and acclimatization. Prior to test start, the test fish were acclimated for one week to the test water and temperature. During holding and acclimatization until one day before the start ofthe test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle 

### I Germany). During the last two weeks prior to the test, no fish died in the test fish batch and all fish were healthy. From the acclimated test fish batch, 10 fish were measured at the start ofthe test. The meanbody length ofthe fish was 2.6 ± 0.05 em (Mean ± SD), the mean body wet weight was 0.11 ± 0.02 g (Mean± SD). The test method and test species are recommended bythe international test guidelines.

Page 12Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

### 3.3 Test Water Report

Reconstituted test water was used in the study. It consisted ofanalytical grade salts dissolved in purified water to obtain the following nominal concentrations: Ingredients I Concentration

Properties mmol/L mg!L CaCh x 2H20 1.0 147 MgS04 x 7H20 0.25 61.5 NaHC03 0.38 32.5 KCl 0.038 2.9 Water Hardness 1.25 125 as CaC03 Alkalinity 0.4 

## --- The ratio ofCa:Mg and Na:K was 4: 1 and 10: 1, respectively, based on molarity. The test water was aerated priorto the preparation ofthe test medium until oxygen saturation was reached. 

### 3.4 Material One glass test vessel with 5 liters oftest medium was used for each treatment. The test vessels were labeled with the study number and all necessary additional information to ensureunique identification. 

### 3.5 Experimental Conditions The water temperature in the test vessels was maintained at 20-22

## oc (Table 5). The test vessels were slightly aerated during the testperiod. A 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period was used (light intensity during the light period was approximately within the range of 100 to 600 Lux). The test duration was 96 hours and the fish were not fed during the test.

Page 13Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

### 3.6 Study Design Report



### A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L. Thus, a single test item concentration with the loading 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 range-finding test (non-GLP). A semi-static test with daily test medium renewal was performed to keep the test item concentrations in the test media as constant as possible during the test period of96 hours. During this semi-static test, the test fish were daily placed into a new test vessel with freshly prepared test medium. The selection ofthe test concentration was based on the results ofarange-finding test (non-GLP). At the start of the test, 7 fish were introduced into each test vessel in a random order. The loading rate was 0.16 g fish wet weight per liter test medium. Thus, the requirement ofa loading rate not exceeding 1 g fish/L was fulfilled. 

### 3.7 Dosage The preparation of the test medium was performed as far as possible in the dark to avoid photolytic degradation ofthe test item. At the start ofthe test and before each test medium renewal, the test medium with the loading rate of 100 mg/L was freshly prepared by completely dissolving 499.98, 499.91, 500.1 and 500.0 mg of the test item in 

### 5 liter of test water, respectively using ultrasonic treatment for 15 minutes and stirring for 96 hours at room temperature in the dark. The long stirring period was chosen in consultation with the sponsor. The test medium was freshly preparedjustbefore start ofthe test and prior to each test medium renewal.

Page 14Harlan Laboratories Study C3ll44 Leuco SulfurBlack I 

### 3.8 Evaluations Report



### 3.8.1 Determination oftheLCSO, LCO, LC100, NOEC and LOEC The test fish were observed for mortality and visible abnormalities after approximately 3, 24, 48, 72 and 96 hours test duration. The NOEC and the LCO were determined directly from the raw data. The LOEC, the LClOO and the LC50 at the observation times could not be quantified due to the absence ofa toxic effect of the testitem at the tested concentration. 

### 3.8.2 Water Quality Criteria The water temperature, pH-values and oxygen concentrations were measured for each treatment at the start ofthe test and once daily during the test in the freshly prepared and old test medium. Atthe same dates the appearance ofthe test medium was recorded. 

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

Page 15Harlan Laboratories Study C31144 Leuco SulfurBlack 1 Report 

## 4 RESULTS AND DISCUSSION A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L. The biological results are listed in Table 1. Inthe control and in the test medium with the loading rate of 100 mg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96-hour NOEC and the 96-hour LCO ofLeuco SulfurBlack 1 to zebra fish were determined to be at the loading rate of least 100 mg/L. The 96-hourNOEC and the 96-hour LCO might even be higher than this loading rate, but loading rates in excess of 100 mg/L have not been tested, according to the guidelines. The 96-hour LOEC, the 96-hour LC50 and the 96-hour LC100 were clearly higher than the loading rate of 100 mg/L. These values could not be quantified due to the absence oftoxicity ofLeuco SulfurBlack 1 at the tested loadingrate. In conclusion, the test item had no acute toxic effects on the test fish up to the loading rate of 100 mg/L. No remarkable observations were made concerning the appearance ofthe test medium. It was a clearthroughout the entire test duration (Table 2). The pH values in the test medium and the control ranged from 7.5 to 7.7 (Table 3). The oxygen concentration was always 8.1 mg/L or higher (Table 4), and thus higher than 60% oxygen saturation. The watertemperature was 20-22

# oc during the test (Table 5).

Page 16Harlan Laboratories Study C31144 Leuco SulfurBlack I 

# 5 REFERENCES Report

1. OECD Series on Testing and AssessmentNo. 23 (2000): Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures

Page 17Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## 6 TABLES Report

Table 1: Mortality and Visible Abnormalities Observed at the TestFish Abbreviations ofvisible abnormalities for which fish were observed duringthe study AK: Strongly extended gills AP: Apathy BA: Distended abdomen BO: Fish mainly atthe bottom ofthe aquarium GA: Exophthalmus KR: Convulsions OB: SA: SR: SV: TS: VF: Fish mainly atthe water surface Mucus secretion Fish lying on side orback on the bottom Strong ventilation Tumbling during swimming Changedbody color
Number offish tested at the single concentration and the control: 

### 7 Loading Number ofabnormal and dead fish I numberofdead fish Type of visible abnormalities rate Observationtime (mg!L) 3hours 24 hours 48 hours 72 hours 96 hours Control 0/0 0/0 0/0 0/0 0/0 100 0/0 0/0 0/0 0/0 0/0 LC50 > 1 00 mg/L basedon the loading rate

Page 18Harlan Laboratories Study C31144 Leuco Sulfur Black 1 Report


### Table 2: Appearance ofthe FreshlyPrepared and Old Test Medium Abbreviations 

### 8: no remarkable observations, clear test medium 9: homogeneous dispersion in the water, turbidity observable 10: noticeable turbidity caused by the test item 11: noticeable coloration causedby the test item 12: inhomogeneous dispersion ofthe test item 13: precipitation ofthe test item 14: test item at the surface 15: test item lying at the bottom ofthe aquarium Exposure time Loading rate 

### Oh 24h 48 h new old new old new old 100 0 0 0 0 0 0 72h 96h new old

0 0

Page 19Harlan Laboratories Study C31144 Leuco SulfurBlack 1 Report
Table 3: pH in the Freshly Prepared and Old Test Medium and in the Control Exposure time Loading rate Oh 24h 48 h 72h new old new old new old new Control 7.6 7.7 7.6 7.6 7.6 7.5 7.6 100 7.6 7.7 7.6 7.6 7.6 7.5 7.6 96h old 7.5 7.6

Table 4: Oxygen Concentrations (mg/L) in the Freshly Prepared and Old Test Medium and in the Control Exposure time Loading rate Oh 24h 48h 72h 96h new old new old new old new old Control 8.6 8.8 8.9 8.7 8.7 8.7 8.5 8.6

100 8.1 8.8 8.3 8.8 8.5 8.6 8.6 8.5

Table 5: Temperatures

# eqin the FreshlyPrepared and Old TestMedium and in the Control Exposure time Loading rate Oh 24h 48 h 72h 96h new old new old new old new old Control 21 21 21 20 21 22 21 22

100 21 21 21 20 21 22 21 21

Page20Harlan Laboratories Study C31144 Leuco SulfurBlack 1 Report



## APPENDIX I- FINALIZED STUDY PLANAND FIRSTAMENDMENT TO STUDYPLAN

Page 21

# harlanTI· STUDYPLAN


## Leuco SulfurBlack 1
Acute Toxicity ofLeuco Sulfur Black 1to ZebraFish (Brachydanio rerio) 

## ina 96-HourTest
StudyDirector: Test Facility:
Sponsor:

StudyIdentification: Version: Dr. A. Peither HarlanLaboratoriesLtd. Zelgliweg 1 44521tingen I Switzerland ClariantProducts (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1 

### I Switzerland

Harlan Laboratories Study C31144 Final
Page 1 of 14 

# ...

Page22Harlan Laboratories Study C31144 Leuco SulfurBlack I 

## CONTENTS Study Plan Page2



### CONTENTS ....................................................................................................................................2 SIGNATURES ................................................................................................................................3 PREFACE .......................................................................................................................................4 General Information...............................................................................................................4 Responsibilities......................................................................................................................4 Schedule.................................................................................................................................4 Good Laboratory Practice......................................................................................................5 Data Requirements ITest Guidelines ....................................................................................5 Animal Welfare......................................................................................................................5 Amendment and Deviation Procedures .................................................................................5 Archiving ...............................................................................................................................6 DEFINITIONS ................................................................................................................................7 1 PURPOSE..............................................................................................................................8 2 MATERIALS AND METHODS...........................................................................................8 2.1 Test ltem..................................................................................................................8 . 2.2 Test System..............................................................................................................9 2.3 TestWater................................................................................................................9 2.4 Material.................................................................................................................. 10 2.5 Experimental Conditions ....................................................................................... 10 2.6 Study Design.......................................................................................................... 10 2.7 Dosage ................................................................................................................... 11 2.8 Evaluations ............................................................................................................ 11 2.8.1 Determination ofthe LC50, NOEC, LOEC, LCO and LC100 ................ 11 2.8.2 Water Quality Criteria ............................................................................. 12 2.8.3 Analysis ofthe Test Item Concentrations ............................................... 12 3 CONDITIONS FORTHE VALIDITY OF THE TEST...................................................... 13 4 REFERENCES .................................................................................................................... 13 5 REPORTING....................................................................................................................... 14 6 DISTRIBUTION ................................................................................................................. 14

Page 23HarlanLaboratories Study C31144 Leuco SulfurBlack l 

## SIGNATURES Study Director:

Management:

Forthe Sponsor: Study Plan Page3



### Dr. A. 

### Peither



# .....tL..**/1~.~······················** Date:

Dr. J. 

### Magyar



# ...

# ~~~i·~·:!l··················

Dr. U. Ensenbach Clariant Produkte (Deutschland) GmbH



# ...«....**~.~!**...............•........... 

### Date: 

# ldld9**~** 

# IJo.,.

# N

Page24Harlan Laboratories Study C31144 Leuco Sulfur Black 1 

## PREFACE General Information Test Item: StudyTitle: Sponsor: StudyMonitor: TestFacility: QA:


### Responsibilities StudyDirector: Deputy StudyDirector: HeadofQA: 

### Schedule Anticipated Experimental Starting Date: Anticipated Experimental CompletionDate: Anticipated Date ofDraftReport: Study Plan Page4

Leuco Sulfur Black 1 Acute toxicity ofLeuco Sulfur Black 1to zebra fish (Brachydanio rerio) in a 96-hour test Clariant Products (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 

### 1 

### I Switzerland Dr. U. Ensenbach HarlanLaboratories 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

Page 25Harlan Laboratories Study C31144 Lcuco Sulfur Black 1 

### Good LaboratoryPractice Study Plan PageS

This study will be performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles ofGood Laboratory Practice, as revised in 1997 and adopted November 261 h, 1997 bydecision 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). 

### Data Requirements 

### ITest Guidelines The study will be performed in accordance with the following internationally accepted guidelines and recommendations: • OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 1992. • EU Commission Directive 92/69/EEC, C.1: Acute Toxicity for Fish, 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.1: Acute Toxicity for Fish. 

### Animal Welfare The study will beperformed in an AAALAC-accredited laboratory in accordance with the Swiss Animal Protection Law under license No. 59 (range finding test) and license No. 60 (definitive test). 

### Amendment and Deviation Procedures Amendments (planned changes) to the studyplan will be issued and signedbythe 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 Sponsor will be promptly informed ofanyrelevant deviations from the studyplan.

Page26Harlan Laboratories Study C31144 Leuco Sulfur Black l 

### Archiving Study Plan Page6



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

Page 27Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## DEFINITIONS LCO: LC50:
LClOO: NOEC (No Observed Effect Concentration): LOEC (1owest Observed Effect Concentration): Treatment: Study Plan Page7

The highest test concentration without a significant number ofdead test organisms. The median lethal concentration, i.e. the calculated concentration oftest item which causes death in 50% of the test organisms. The lowest test concentration at which all test organisms are dead. The highest test concentration at which no significant toxic effect on the test organisms is observed. The lowest test concentration at which a significant toxic effect on the test organisms is observed. Comprises test item treatments, control (test medium only) and solvent control (ifapplicable).

Page 28Harlan Laboratories Study C31144 Leuco SulfurBlack I 

## 1 PURPOSE Study Plan Page 8

The purpose of this 96-hour toxicity test is to evaluate the acute toxicity of the test item Leuco Sulfur Black 1 to fish. Zebra fish 

### (Brachydanio rerio) will be exposed to aqueous test media containing the test item at a range ofdifferent concentrations under defined conditions. Recorded effects will bemortality and visible abnormalities ofthe test fish. 

## 2 MATERIALS AND METHODS 2.1 Test Item The test item and the following information were provided bythe Sponsor: Identity: CAS No.: BatchNo.: Description: Expiration date: Composition:

Purity: Stability: Storage conditions:
Safetyprecautions: Leuco Sulfur Black 1 66241-11-0 ESA0007595 Thixotropic, black, wetpaste 11-July-2011

I. Leuco Sulfur Black 1: 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 Atroom temperature (range 20 +/- 5°C), light protected. At Harlan Laboratories: atroom temperature, away from direct sunlight Routine hygienic procedures are sufficient to ensure personnel health and safety.

Page 29Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

### 2.2 Test System Study Plan Page9

The study will be performed with zebra fish (Brachydanio rerio). The fish used for the test will be obtained from a commercial supplier. In accordance with the test guidelines, the fish will be held at Harlan Laboratories for at least twelve days. Alternatively, the fish may be obtained from a breeding culture at Harlan Laboratories Ltd. The fish will be acclimated to the test water and test temperature for at least seven days prior to test start. During holding, the fish will be fed with a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle I Germany) until one day prior to test start. The body wet weight and body length ofat least ten fish ofthe acclimated test fish batch will be determined prior to test start. The mean body length ofthe test fish will be about 3.0 ± 0.5 em. The measured fish will notbeused for the test. The test method and the test species are recommended bythe international test guidelines. 

### 2.3 Test Water Reconstituted test water will be used in the study. It will consist of analytical grade salts dissolved in purified waterto obtain the following nominal concentrations: Ingredients I Concentration

Properties mmol/L mg/L CaCh x 2H20 1.0 147 MgS04 X 7H20 0.25 61.5 NaHC03 0.38 32.5 KCl 0.038 2.9 Water Hardness 1.25 125 as CaC03 Alkalinity 0.4 

## --- The ratio ofCa:Mg and Na:K will be4:1 and 10:1, respectively, based on molarity. The test water will be aerated prior to the preparation ofthe test media until oxygen saturation is reached. The pH ofthe reconstituted water after equilibration will be in the range of6.0 to 8.5. Alternatively, local tap water (non chlorinated well water ofdrinking water quality), reduced for total hardness by ion exchange will be used. The total hardness will be about 200 to 250 mg/L CaC03. Analyses will be given in the report. This test water will be aerated prior to the preparation ofthe test media until oxygen saturation is reached. The pH ofthe local tap water will be in the range of7.8 to 8.8.

Page 30Harlan Laboratories Study C31144 Leuco Sulfur Black 1 

### 2.4 Material Study Plan Page 10

One test vessel with at least two liters test medium will be used per treatment. The test vessels will be labeled with the study number and all necessary additional information to ensure unique identification. 

### 2.5 Experimental Conditions The water temperature in the test vessels will be maintained at 21-24 °C 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 lightperiod will be between 50 and 600 Lux. The test duration will be 96 hours. 

### 2.6 Study Design At the start ofthe test, seven fish will be introduced into each test vessel in a random order. The loading rate will not exceed 1 g fish wet weight per liter medium. The fish will not be fed during the test. Ifnecessary, the testmedia will be slightly aerated during the test. A semi-static test with daily test medium renewal will be performed to keep the test item concentrations in the test media as constant as possible during the test period of96 hours. During this semi-static test, the surviving fish will be transferred daily into a new test vessel with freshly prepared test medium ofthe corresponding treatment. A geometric series ofat least five concentrations differing by a constant factor not exceeding 2.2 will be used. The chosen test concentrations should permit the determination ofthe LC50 and, if possible, the LCO and LC100. Concentrations ofthe 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 beused for other studies running inparallel atHarlan Laboratories. 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 Page 31Harlan Laboratories Study C31144 Leuco SulfurBlack 1 Study Plan Page 11
final 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 order to assess its toxicity, water accommodated fractions (WAFs) will be prepared. Individual dispersions ofthe test item will bepreparedby 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/orintense stirring. · The dispersions will be stirred on magnetic stirrers atroom 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 providedbythe sponsor). After stirring, dispersions will be filtered through membrane filters (Schleicher & Schuell, Type NC45, pore size 0.45**~m)** just before the start of the test. The filtrates of the equilibrated dispersionswill betested-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 thepreparation ofthe WAFs. The preparation ofthe test media will be based on the OECD Guidance Document on Aquatic Toxicity Testing ofDifficult Substances andMixtures [1]. 

### 2.8 Evaluations 2.8.1 Determination ofthe LCSO, NOEC,LOEC, LCO and LClOO The fish will be checked about 2-4, 24, 48, 72 and 96 hours after test start for visible abnormalities and mortality (observations will beperformed before the test medium renewal). If applicable, the LC50 and 95% confidence limits for each time point will be calculated by appropriate statistical tests as e.g., Probit Analysis [2, 3] orMoving Average Interpolation [4, 5]. Ifpossible, the NOEC, LOEC, LCO and LClOO will be determined directly from the raw data without any data transformation.

Page 32Harlan Laboratories Study C31144 Leuco SulfurBlack I 

### 2.8.2 Water Quality Criteria Study Plan Page 12

The pH-values, oxygen concentrations and water temperature will be measured in each test medium and the control prior to the introduction ofthe fish. Thereafter, these parameters will be measured at least once every day during the test in the freshly prepared and aged test media. At the same time, the appearance ofthe test media will be recorded. The pH, oxygen concentration and water temperature will be measured in each test medium and the control prior to the introduction ofthe fish, and thereafter at least once every day during the test. The appearance ofthe test media will berecorded at least daily. 

### 2.8.3 Analysis ofthe Test Item Concentrations Onrequest ofthe sponsor, no analytical work will be performed.

Page 33Harlan Laboratories Study C31144 Leuco SulfurBlack 1 Study Plan 

## 3 CONDITIONS FOR THE VALIDITY OF THE TEST A test is not acceptable ifmore than one control fish dies during the 96-hourtestperiod. Page 13

The oxygen concentration in the test media should not drop below 60% of the air saturation during the test. 

## 4 REFERENCES 1. OECD Series on Testing and AssessmentNo. 23 (2000): Guidance Document on Aquatic ToxicityTesting ofDifficult Substances and Mixtures 2. FINNEY, D.J. (1971): Probit Analysis, 3rd Edition, Cambridge University Press, London 3. DAVIES, R.G. (1971): Computer Programming in Quantitative Biology, Academic Press, London 4. THOMPSON, W.R., WElL, C.S. (1952): On the construction oftables for moving average interpolation, Biometrics 8, 51-54 5. FINNEY, D.J. (1978): Statistical Methods in Biological Assay, 3rdEdition, Charles Griffin, London

Page 34Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## 5 REPORTING Study Plan Page 14

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 beissued. 

## 6 DISTRIBUTION This study plan will be distributed as follows: Sponsor Monitor 

### I Sponsor'srepresentative: Harlan Laboratories Ltd. StudyFile: 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 35

## FIRSTAMENDMENT TO STUDYPLAN


## Leuco SulfurBlack 1
Acute Toxicity ofLeuco SulfurBlack 1to Zebra Fish (Brachydanio rerio) 

## ina 96-HourTest
StudyDirector: TestFacility:
Sponsor:

StudyIdentification: Version: Dr. A. Peither 

### HarlanLaboratories Ltd. Zelgliweg 1 4452 Itingen 

### I Switzerland 

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

### I Switzerland

HarlanLaboratories Study C31144 Final
Page 1of5 

# haria

# nne...

Page 36Harlan Laboratories Study C31144 First Amendmentto Study Plan Page2 Lcuco SulfurBlack I · 

## SIGNATURES Study 

### Director: Dr. A. Peither

# d4·~~ .................................................. Date:
For the Sponsor: Dr. U. Ensenbach Clariant

### Products (Switzerland) Ltd.



# ...f.;...**~~**

# ....... Date:

## ~ 

# ?Ji**;'~**

# f

Page 37Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## PAGE 8 

## CONCERNING FirstAmendment to Study Plan Page3



### 2 MATERIALS AND METHODS 2.1 Test Item 

## PRESENT Identity: CAS No.: Batch No.: Description: Expiration date: Composition:

Purity: Stability: Storage conditions:
Safetyprecautions: Leuco SulfurBlack 1 66241-11-0 ESA0007595 Thixotropic, black, wetpaste 11-July-2011

I. Leuco Sulfur Black 1: 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. Stableunder storage conditions Atroom 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 38Harlan Laboratories Study C31144 Leuco SulfurBlack 1 

## NEW Identity: CAS No.: Description: BatchNo.: Composition:

Stability ofTestItem: Expiry Date: Storage Conditions: Safetyprecautions: First Amendmentto Study Plan

Leuco Sulfur Black 1 66241-11-0 Thixotrophic, black, wetpaste ESA0007595 Page4

Leuco Sulfur Black 1: 52.75% (calculatedbyamount of by-compounds) 2,4-Dinitrochlorobenzene: <20 ppm 2,4-Dinitrophenole: <20ppm Sulfate: 970 ppm Chloride: 750 ppm Sodium: 1.3% (w/w) Water: 45.75% (w/w) Stable under storage conditions 11-Jul-2011 At room temperature (range 20 ± 5°C), lightprotected. Routine hygienic procedures are sufficient to ensure personnel health and safety. 

## REASON FORTHEALTERATION Standardizedtest item information requested by the Sponsor.

•**~_**__**i~.~** :·•. _,'r:<Dd1ltf Page 39Harlan Laboratories Study C3I I44 Leuco SulfurBlack 1 

## DISTRIBUTION FirstAmendmentto StudyPlan

This studyplan amendment will be distributed as follows: Sponsor Monitor 

### I Sponsor's representative: Harlan Laboratories Ltd. StudyFile: StudyDirector: Technical Coordinator: Logistics: QAESM: 1 x copy
1 x original 1 x copy 1 x copy 1 x copy 1 x copy PageS

Page40