DISCLAIMER 

This report is the property of Clariant. 

Its content is confidential and not for general 

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

or its content to a third party without prior written 

approval of Clariant . 

REPORT 

Leuco Sulfur Black 1 

Acute Toxicity ofLeuco Sulfur Black 1 to Zebra Fish 

(Brachydanio rerio) in a 96-Hour Test 

Study Director: 

Test Facility: 

Sponsor: 

Dr. A. Peither 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 Itingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Study Identification: Harlan Laboratories Study C31144 

Version: Final 

Study Completion Date: 07-Aug-2009 

Page 1 of40 

CONTAINS NO CBt 

('-PH~ 

" r:;_1; l--:2 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 

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

Animal Welfare ...................................................................................................................... 8 

Summary of Study Plan 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 Test Water .............................................................................................................. 13 

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

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

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

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

3.8 Evaluations ............................................................................................................ 15 

3.8.1 Determination of the LC50, LCO, LClOO, NOEC and LOEC ................ 15 

3.8.2 Water Quality Criteria ............................................................................. 15 

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

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

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

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

APPENDIX I- FINALIZED STUDY PLAN AND FIRST AMENDMENT TO 

STUDY PLAN ..................................................................................................................... 21 

Page2 
Harlan Laboratories Study C31144 

Leuco Sulfur Black I 

Report 

LIST OF TABLES 

Table 1: Mortality and Visible Abnormalities Observed at the Test Fish ................................ 18 

Table 2: Appearance of the Freshly Prepared and Old Test Medium ....................................... 19 

Table 3: pH in the Freshly Prepared and Old Test Medium and in the Control ....................... 20 

Table 4: Oxygen Concentrations (mg/L} in the Freshly Prepared and Old Test 

Medium and in the Control ......................................................................................... 20 

Table 5: Temperatures ec) in the Freshly Prepared and Old Test Medium and 

in the Control .............................................................................................................. 20 

Page 3 
Harlan Laboratories Study C31144 

Leuco Sulfur Black l 

GLP CERTIFICATE 

11'11 Schwelzerlsche lldgenossenschift 

y Conf!ch!ration suisse 

Confederazlone Svh:zera 

Conftderazlun svixra 

Report 

Federal Department of Homt Affairs DHA 

Pederal Offlce of Public Hnhh POPH 

ftdtrallnpartmtnt of the Environment. 

Transport. Energy and Communlcaclons DETEC 

Peden~ Oftke for tht !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 

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

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

Laboratory Pracllce, as revised In 1997 and adopted on 26th November 1997 by decision of the 

OECD Council [C(97)1861Final]. 

Unequivocal name and address 

of the test facility: 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen, Switzelland. 

Areas of expertise according to 

article 3 paragraph 1 Jetter d OGLP: 

1 J 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./ Residue studies, 

7 .I Studies on effects on mesocosms and 

natural ecosystems, 

8.1 Analytical and clinical chemistry testing, 

9./ Other studies (safety pharmacology and 

animal metabolism). 

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

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

Data of Inspection: 05th to 09th and 26th to 3oth November 2007 

Date of decision: 30th April 2008 

Based on the above mentioned decision H can 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. 

1ht noc11c1t1Dn aultlodlyfotehllnlelfS Ia INI c:oan:lnllllon and decWon auttol)'far .. good labonllory pracllct (Gl.P) far h FOEN, .. FOPH llld Swlamedlc. 

8Mn Fldatal Qmca at P~Htlllh. Conunerprotectlonclnlct:cnle, H~aullodtyfarc:hlalll*s, CH.aoD3 lem. 

w.mplp·!dm"'!!h: PhDn« <t41 ((!)313227305 Fax: +41 f013132354118 

Page4 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

GOOD LABORATORY PRACTICE 

Report 

STATEMENT OF COMPLIANCE 

Harlan Laboratories Study: 

Test Item: 

Study Director: 

Study Title: 

C31144 

Leuco Sulfur Black 1 

Dr. A. Peither 

Acute toxicity of Leuco Sulfur Black 1 to 

zebra fish (Brachydanio rerio) in a 96-hour test 

This study has been performed in compliance witli the Swiss Ordinance relating to Good 

Laboratory Practice, adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the 

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

\ 

1997 by decision of the OECD Council [C(97) 186/Final). 

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHL W, MAFF and MET!). 

Exclusions: 

- Pre-experiments as mentioned in the report. 

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

Study Director: Dr. A. Peither 

.. i .. t.~~ ........................ 

Date: fot 3 ~ 1, 2. () () <( 

Page 5 
Harlan Laboratories Study C31144 

Leuco Sulfur Black I 

Report 

QUALITY ASSURANCE GLP STATEMENT 

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

Harlan Laboratories Study: 

Test Item: 

Study Director: 

C31144 

Leuco Sulfur Black 1 

Dr. A. Peither 

Study Title: Acute toxicity of Leuco Sulfur Black 1 to 

zebra fish (Brachydanio rerio) in a 96-hour test 

The general facilities and activities are inspected at least once a year and the results are reported 

to the responsible person and the management. 

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

Quality Assurance. The dates are given below. 

Dates of Reports 

Dates and Types of QA Inspections to the Study Director 

and to the Test Facility 

Management 

20-Apr-2009 Study plan 20-Apr-2009 

20-Apr-2009 Process based (test conditions) 20-Apr-2009 

26-Jun-2009 Report 26-Jun-2009 

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

Quality Assurance: Ms. S. Zeugin 

___s.. 27'"" ····························································· 

Page 6 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

HeadofQA: 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Report 

Leuco Sulfur Black 1 

Acute toxicity of Leuco Sulfur Black 1 to 

zebra fish (Brachydanio rerio) in a 96-hour 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 Itingen I Switzerland 

Dr. A. Peither 

Dr. S. Hoger 

Mr. T. Fink 

04-May-2009 

15-May-2009 

Page? 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

Report 

Data Requirements I Test Guidelines 

This study followed the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

• OECD Guideline for 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 of 30 May 2008 laying down test methods 

pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the 

Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals 

(REACH), C.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 of Study Plan Amendments 

First Amendment to Study Plan 

Concerning: Alteration: Reason: 

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

Sponsor 

-Reanalysis of test item composition 

Archiving 

Harlan Laboratories Ltd. ( 4452 ltingen/Switzerland) will retain the study plan, raw data, a 

sample of the test item and the final report of the present study for at least ten years. No data will 

be discarded without the Sponsor's written consent. 

Page 8 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 of dead test organisms. 

The median lethal concentration, i.e. the calculated 

concentration of test 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 (if applicable). 

Page 9 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

1 SUMMARY 

Report 

The acute toxicity of the 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 

Chemicals No. 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 of the test item, the test medium was prepared prior to the start of 

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

loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 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 of the test medium was based on the OECD Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures, 2000 [1]. 

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

water). 

The biological results were based on the loading rate of the test item. 

In the control and at the loading rate of the test item of 100 mg/L no mortality or other visible 

abnormalities were determined during the test period of 96 hours. 

The summary of the biological results is as follows: 

- 96-hour LC50: 

- 96-hour LCO: 

- 96-hour LClOO: 

- 96-hour NOEC: 

- 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 10 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

2 PURPOSE 

Report 

The purpose of this 96-hour toxicity test was to evaluate the acute toxicity of the test item 

Leuco Sulfur Black 1 to 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 11 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 of Test Item: 

Expiry Date: 

Storage Conditions: 

3.2 Test System 

Leuco Sulfur Black 1 

66241-11-0 

Thixotrophic, black, wet paste 

ESA0007595 

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

by-compounds) 

2,4-Dinitrochlorobenzene: <20 ppm 

2,4-Dinitrophenole: <20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water: 45.75% (w/w) 

Stable under storage conditions 

11-Jul-2011 

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

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 of the 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 of the test. The mean body 

length of the 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 by the international test guidelines. 

Page 12 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

3.3 Test Water 

Report 

Reconstituted test water was used in the study. It consisted of analytical 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 of Ca:Mg and N a:K was 4: 1 and 10: 1, respectively, based on molarity. The test water 

was aerated prior to the preparation of the test medium until oxygen saturation was reached. 

3.4 Material 

One glass test vessel with 5 liters of test medium was used for each treatment. 

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

ensure unique 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 test period. 

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 13 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 of the test item). The limit test was based on the results of a 

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 of 96 hours. During 

this semi-static test, the test fish were daily placed into a new test vessel with freshly prepared 

test medium. 

The selection of the test concentration was based on the results of a range-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 of a 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 of the test item. 

At the start of the 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 prepared just before start of the test and prior to each test medium 

renewal. 

Page 14 
Harlan Laboratories Study C3ll44 

Leuco Sulfur Black I 

3.8 Evaluations 

Report 

3.8.1 Determination ofthe LCSO, 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 of a toxic effect of 

the test item 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 of the test and once daily during the test in the freshly prepared and old test medium. 

At the same dates the appearance of the test medium was recorded. 

3.8.3 Analysis of the Test Item Concentrations 

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

water). 

Page 15 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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. In the 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 of Leuco Sulfur Black 1 to zebra fish were 

determined to be at the loading rate of least 100 mg/L. The 96-hour NOEC 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 of toxicity of Leuco Sulfur Black 1 at the tested loading rate. 

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 of the test medium. It was a 

clear throughout 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 water temperature was 20-22 oc during the test (Table 5). 

Page 16 
Harlan Laboratories Study C31144 

Leuco Sulfur Black I 

5 REFERENCES 

Report 

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

Guidance Document on Aquatic Toxicity Testing 

of Difficult Substances and Mixtures 

Page 17 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

6 TABLES 

Report 

Table 1: Mortality and Visible Abnormalities Observed at the Test Fish 

Abbreviations of visible abnormalities for which fish were observed during the study 

AK: Strongly extended gills 

AP: Apathy 

BA: Distended abdomen 

BO: Fish mainly at the bottom 

of the aquarium 

GA: Exophthalmus 

KR: Convulsions 

OB: 

SA: 

SR: 

SV: 

TS: 

VF: 

Fish mainly at the water surface 

Mucus secretion 

Fish lying on side or back on the bottom 

Strong ventilation 

Tumbling during swimming 

Changed body color 

Number of fish tested at the single concentration and the control: 7 

Loading Number of abnormal and dead fish I number of dead fish 

Type of visible abnormalities 

rate 

Observation time 

(mg!L) 3 hours 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 based on the loading rate 

Page 18 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

Report 

Table 2: Appearance of the Freshly Prepared 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 caused by the test item 

12: inhomogeneous dispersion of the test item 

13: precipitation of the test item 

14: test item at the surface 

15: test item lying at the bottom of the 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 19 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 eq in the Freshly Prepared and Old Test Medium 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 

Page20 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

Report 

APPENDIX I- FINALIZED STUDY PLAN AND 

FIRST AMENDMENT TO STUDY PLAN 

Page 21 
harlanTI· 

STUDY PLAN 

Leuco Sulfur Black 1 

Acute Toxicity ofLeuco Sulfur Black 1 to Zebra Fish 

(Brachydanio rerio) in a 96-Hour Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

Version: 

Dr. A. Peither 

Harlan Laboratories Ltd. 

Zelgliweg 1 

44521tingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Harlan Laboratories Study C31144 

Final 

Page 1 of 14 

... 

Page22 
Harlan Laboratories Study C31144 

Leuco Sulfur Black I 

CONTENTS 

Study Plan Page2 

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

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

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

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

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

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

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

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

Animal Welfare ...................................................................................................................... 5 

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

Archiving ............................................................................................................................... 6 

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 of the LC50, NOEC, LOEC, LCO and LC100 ................ 11 

2.8.2 Water Quality Criteria ............................................................................. 12 

2.8.3 Analysis of the Test Item Concentrations ............................................... 12 

3 CONDITIONS FOR THE VALIDITY OF THE TEST ...................................................... 13 

4 REFERENCES .................................................................................................................... 13 

5 REPORTING ....................................................................................................................... 14 

6 DISTRIBUTION ................................................................................................................. 14 

Page 23 
Harlan Laboratories Study C31144 

Leuco Sulfur Black l 

SIGNATURES 

Study Director: 

Management: 

For the Sponsor: 

Study Plan Page3 

Dr. A. Peither 

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

Dr. J. Magyar 

... ~~~i·~·:!l·················· 

Dr. U. Ensenbach 

Clariant Produkte (Deutschland) GmbH 

... «.. .. ~.~! ...............•........... 

Date: ldld9 ~ IJo.,. N 

Page24 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

HeadofQA: 

Schedule 

Anticipated Experimental 

Starting Date: 

Anticipated Experimental 

Completion Date: 

Anticipated Date of Draft Report: 

Study Plan Page4 

Leuco Sulfur Black 1 

Acute toxicity of Leuco Sulfur Black 1 to zebra fish 

(Brachydanio rerio) in a 96-hour test 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 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 

Page 25 
Harlan Laboratories Study C31144 

Lcuco Sulfur Black 1 

Good Laboratory Practice 

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

h, 

1997 by decision of the OECD Council [C(97)186/Final]. 

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHLW, MAFF and METI). 

Data Requirements I Test 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 of 30 May 2008 laying down test methods 

pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the 

Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals 

(REACH), C.1: Acute Toxicity for Fish. 

Animal Welfare 

The study will be 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). 

Amendment and Deviation Procedures 

Amendments (planned changes) to the study plan will be issued and signed by the Study Director, 

and will become effective at the time of the Study Director's signature. The Sponsor will receive 

the original amendment, which must be signed and returned to Harlan Laboratories Ltd., and one 

copy. The amendment will be distributed (see Distribution) and added to all copies of the study 

plan. 

Deviations (unplanned changes) to the study plan will be documented and acknowledged by the 

Study Director and maintained with the raw data. The report will reflect any deviations. The 

Sponsor will be promptly informed of any relevant deviations from the study plan. 

Page26 
Harlan 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 of the test item and the final report of the present study for at least ten years. No data will 

be discarded without the Sponsor's written consent. 

Page 27 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 of dead test organisms. 

The median lethal concentration, i.e. the calculated 

concentration of test 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 (if applicable). 

Page 28 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 of different concentrations under defined conditions. 

Recorded effects will be mortality and visible abnormalities of the test fish. 

2 MATERIALS AND METHODS 

2.1 Test Item 

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

Identity: 

CAS No.: 

Batch No.: 

Description: 

Expiration date: 

Composition: 

Purity: 

Stability: 

Storage conditions: 

Safety precautions: 

Leuco Sulfur Black 1 

66241-11-0 

ESA0007595 

Thixotropic, black, wet paste 

11-July-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. 

Stable under storage conditions 

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

protected. 

At Harlan Laboratories: at room temperature, away 

from direct sunlight 

Routine hygienic procedures are sufficient to ensure 

personnel health and safety. 

Page 29 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 of at least ten fish of the acclimated test fish batch will be 

determined prior to test start. The mean body length of the test fish will be about 3.0 ± 0.5 em. 

The measured fish will not be used for the test. 

The test method and the test species are recommended by the 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 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 will be 4:1 and 10:1, respectively, based on molarity. 

The test water will be aerated prior to the preparation of the test media until oxygen saturation is 

reached. The pH of the reconstituted water after equilibration will be in the range of 6.0 to 8.5. 

Alternatively, local tap water (non chlorinated well water of drinking 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 of the test media until oxygen saturation is reached. The pH of the local tap water 

will be in the range of7.8 to 8.8. 

Page 30 
Harlan 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 light period will be between 50 and 600 Lux. 

The test duration will be 96 hours. 

2.6 Study Design 

At the start of the 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. If necessary, the test media 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 of 96 hours. During 

this semi-static test, the surviving fish will be transferred daily into a new test vessel with freshly 

prepared test medium of the corresponding treatment. 

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

will be used. The chosen test concentrations should permit the determination of the LC50 and, if 

possible, the LCO and LC100. Concentrations of the test item higher than 100 mg/L or above the 

solubility limit of the test item in the test water (whichever is the lower) will usually not be 

tested in accordance with the test guidelines. 

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

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

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

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

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

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

test (Limit Test according to the test guidelines). 

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

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

Page 31 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

Study Plan Page 11 

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

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

number of the present study. 

2.7 Dosage 

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

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

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

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

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

and/or intense stirring. · 

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

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

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

provided by the sponsor). 

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

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

dispersions will be tested-as WAFs. 

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

renewal. 

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

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

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

Toxicity Testing of Difficult Substances and Mixtures [1]. 

2.8 Evaluations 

2.8.1 Determination of the 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 be performed 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] or Moving Average Interpolation [4, 5]. 

If possible, the NOEC, LOEC, LCO and LClOO will be determined directly from the raw data 

without any data transformation. 

Page 32 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 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 of the 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 of the 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 of the fish, and thereafter at least once every day during the 

test. The appearance of the test media will be recorded at least daily. 

2.8.3 Analysis of the Test Item Concentrations 

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

Page 33 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

Study Plan 

3 CONDITIONS FOR THE VALIDITY OF THE TEST 

A test is not acceptable if more than one control fish dies during the 96-hour test period. 

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 Assessment No. 23 (2000): 

Guidance Document on Aquatic Toxicity Testing 

of Difficult 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 of tables for moving average interpolation, 

Biometrics 8, 51-54 

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

Statistical Methods in Biological Assay, 

3rd Edition, Charles Griffin, London 

Page 34 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

5 REPORTING 

Study Plan Page 14 

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

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

6 DISTRIBUTION 

This study plan will be distributed as follows: 

Sponsor 

Monitor I Sponsor's representative: 

Harlan Laboratories Ltd. 

Study File: 

Study Director: 

Technical Coordinator: 

Logistics: 

QAESM: 

1 x copy 

1 x original 

1 x copy 

1 x copy 

1 x copy 

1 x copy 

Page 35 
FIRST AMENDMENT TO STUDY PLAN 

Leuco Sulfur Black 1 

Acute Toxicity ofLeuco Sulfur Black 1 to Zebra Fish 

(Brachydanio rerio) in a 96-Hour Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

Version: 

Dr. A. Peither 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 Itingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Harlan Laboratories Study C31144 

Final 

Page 1 of5 

haria nne... 

Page 36 
Harlan Laboratories Study C31144 First Amendment to Study Plan Page2 

Lcuco Sulfur Black I · 

SIGNATURES 

Study Director: Dr. A. Peither 

.................................................. d4·~~ Date: 

For the Sponsor: Dr. U. Ensenbach 

Clariant Products (Switzerland) Ltd . 

... f.; ... ~~ ....... Date: ~ ?Ji ;'~ f 

Page 37 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

PAGE 

8 

CONCERNING 

First Amendment 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: 

Safety precautions: 

Leuco Sulfur Black 1 

66241-11-0 

ESA0007595 

Thixotropic, black, wet paste 

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. 

Stable under storage conditions 

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

protected. 

At Harlan Laboratories: at room temperature, away 

from direct sunlight 

Routine hygienic procedures are sufficient to ensure 

personnel health and safety. 

Page 38 
Harlan Laboratories Study C31144 

Leuco Sulfur Black 1 

NEW 

Identity: 

CAS No.: 

Description: 

Batch No.: 

Composition: 

Stability of Test Item: 

Expiry Date: 

Storage Conditions: 

Safety precautions: 

First Amendment to Study Plan 

Leuco Sulfur Black 1 

66241-11-0 

Thixotrophic, black, wet paste 

ESA0007595 

Page4 

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

by-compounds) 

2,4-Dinitrochlorobenzene: <20 ppm 

2,4-Dinitrophenole: <20 ppm 

Sulfate: 970 ppm 

Chloride: 750 ppm 

Sodium: 1.3% (w/w) 

Water: 45.75% (w/w) 

Stable under storage conditions 

11-Jul-2011 

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

Routine hygienic procedures are sufficient to ensure 

personnel health and safety. 

REASON FOR THE ALTERATION 

Standardized test item information requested by the Sponsor . 

• ~_ __ i~.~ :· •. 

_,'r:<Dd1ltf Page 39 
Harlan Laboratories Study C3 I I 44 

Leuco Sulfur Black 1 

DISTRIBUTION 

First Amendment to Study Plan 

This study plan amendment 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 

PageS 

Page40