### DISCLAIMER This report is the property of 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 of Clariant . 

## REPORT Leuco SulfurBlack 1 Ready Biodegradability ofLeuco SulfurBlack 1 in a Manometric Respirometry Test
StudyDirector: TestFacility:
Sponsor: Dr. U. Memmert Harlan Laboratories Ltd. Zelgliweg 1 4452 ltingen I 

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

### Switzerland StudyIdentification: Harlan Laboratories Study C31177 Version: Final StudyCompletion Date: 19-Aug-2009 Page 1of45


## CONTAtNS NO CBt 

# harianTh 

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# bp1/ I""J-Harlan Laboratories Study C31177 Leuco Sulfur Black I 

## CONTENTS Report



### CONTENTS ....................................................................................................................................2 LIST OF TABLES ..........................................................................................................................3 LIST OF FIGURES......................................................................................................................... 3 GLP CERTIFICATE.......................................................................................................................4 STATEMENT OF COMPLIANCE................................................................................................5 QUALITY ASSURANCE GLP STATEMENT.............................................................................6 PREFACE .......................................................................................................................................7 General Information...............................................................................................................7 Responsibilities......................................................................................................................7 Schedule.................................................................................................................................7 Data Requirements I

### Test Guidelines .................................................................................... 8 Summary ofStudy 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 Reference Item....................................................................................................... 12 3.3 Test System............................................................................................................ 13 3.4 Test Procedures...................................................................................................... 13 3.4.1 Test Water ............................................................................................... 13 3.4.2 Test Concentrations................................................................................. 14 3.4.3 Test Conditions........................................................................................ 15 3.5 Calculations ........................................................................................................... 16 3.6 Determination ofthe Chemical Oxygen Demand ................................................. 16 4 RESULTS AND DISCUSSION.......................................................................................... 17 4.1 Biodegradation ofthe Test Item ............................................................................ 17 4.2 Biodegradation ofthe Reference Item................................................................... 17 4.3 Biodegradation in the Toxicity Control................................................................. 18 4.4 Chemical Oxygen Demand (COD) ofthe Test Item ............................................. 18 4.5 pH Measurement.................................................................................................... 18 5 TABLES .............................................................................................................................. 19 6 FIGURES.............................................................................................................................22 APPENDIX I- FINALIZED STUDY PLAN AND FIRST AMENDMENT TO STUDY PLAN.....................................................................................................................24

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# r**4.~.~\--l',** -tj..A• ·,.**',:;~,_:·n;:•** Page 2Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## LIST OF TABLES Report

Table 1 Oxygen Consumption in the TestFlasks .................................................................... 19 Table 2 Biodegradation in the Test Flasks...............................................................................20 Table 3 pH Values at the Start and at the End ofthe Test.......................................................21 

## LIST OFFIGURES Figure 1 Figure 2 Oxygen Consumption in the Test Flasks during the Incubation Period .....................22 Biodegradation in the Test Flasks during the Incubation Period................................23

Page 3Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## GLP CERTIFICATE

Schweizerische**Eidgenossensch~ft Conf~d~ration**  suisse Confederazione Svizzera Conflderniun svizra Swiss Confederation Report

The SwinGlPMonlkrtlgAuthortlt• Federal Department of Home Affaln: DHA**Peden~** Offlca of Public Hftlth POPH Federal Department of the Environment, Transport. Energy and Communications DETEC Feder-.1 Office forthe: !nvlronment PO!N 

# / sw1ssmedic 

## Statement ofGLP Compliance According to Article 14 paragraph 3 Ordinance on Good Laboratory Practice [OGLP, SR 813.112.1] The noti!Jcatlon authority for chemicals confirms that the following test facility was inspected with respect to the compliance with the SWiss Ordinance on Good Laboratory Practice, adopted on 18th May 2005 [OGLP, SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised In 1997 and adopted on 26th November 1997 by decision of the OECD Councii[C(97)1861Final]. Unequivocal name and address ofthe test facility: Areas ofexpertise according to article 3 paragraph 1 letter d OGLP: Har1an Laboratories Ltd. Zelgliweg 1 4452 Wngen, Switzer1and. 1JPhysical-chemical testing, 2.1 Toxicity studies, 4.1 Environmental toxicity studies on aquatic and terrestrial organisms, 5.1 Studies on behaviour in water, soil and air; bioaocumulation, 6.1 Residue studies, 7.1 Studies on effects on mesocosms and natural ecosystems, 8./Analytical and clinical chemistry testing, 9./Other studies (safety pharmacology and animal metabolism). Inspection authority: Federal Office for the Environment (FOEN) I Federal Office of Public Health (FOPH) ISwissAgency forTherapeutic Products (Swissmedic) Date of inspection: 05th to 09th and 26th to 3oth November2007 Date ofdecision: 30th April 2008 Based on the above mentioned decision It can be confirmed that the above mentioned test facility is able to conduct studies according to the aforementioned areas of expertise in compliance with the principles of GLP. The above mentioned test facility 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. Swiss**Fede~l,\)ff!ce** of Public Health Consumer protection directorate

llwnotilcetlcnauhlrityfOrehemlella IsfieCOOI'tiiM!iOn anddadllon**~for** lhl good llboralory prdc.(GLPI far theFOEN, lhe FOPHand 8wl:umec:llc. tMtu Fad'..,O!nDe cAPt.tlllcHealth, Con1uMtpn;Mttiond.-.ct«ate, No!lllcallon IWihorilyforc;:temicala, CH-3003 Sem. ""'*EmDen Phone: +41l0)313227305 Fax: +41 (0)3132354 8S

Page4Harlan Laboratories Study C31177 Leuco SulfurBlack 1 GOOD LADORATORY PRACTICE Report



## STATEMENT OF COMPLIANCE Harlan Laboratories Study: Test Item: Study Director: C31177 Leuco SulfurBlack I Dr. U. Memmert Study Title: Ready Biodegradability ofLeuco SulfurBlack l in a Manometric Respirometry Test This study has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted May 181 h, 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), andJapan (MHLW, MAFF and MET!). Exclusions: Solubility pre-experiment ofthe test item in test water. Determination ofthe COD ofthe test item. Rationale for selection ofBMG Engineering AG for COD determination: The COD (Chemical Oxygen Demand) of the test item was not determined at Harlan Laboratories Ltd. This work was subcontracted to BMG Engineering AG, 8952 Schlieren I Switzerland, a company certified by the Swiss Testing Service to perform the COD determination according to ISO/IEC 17025. As the COD determination was not performed under GLP, it is excluded from the Statement ofCompliance. There were no circumstances that may have affected the quality or integrity ofthe data. StudyDirector: Dr. U. Memmert



# ......!!M:Jt......................**Date:~~~**

Page 5Harlan Laboratories Study C31177 Leuco SulfurBlack 1 Report 

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

Harlan Laboratories Study: Test Item: Study Director: C31177 Leuco Sulfur Black 1 Dr. U. Memmert Study Title: ReadyBiodegradability ofLeuco SulfurBlack 1 in a Manometric Respirometry Test The general facilities and activities are inspected at least once a year and the results are reported to the responsible person andthe management. Study procedures (with the exception of the COD determination of the test item) were periodically inspected. The study plan and this report were audited by the Quality Assurance. The dates are given below. Dates ofReports Dates and Types ofQA Inspections to the StudyDirector and totheTestFacility Management 05-Mar-2009 Study Plan 05-Mar-2009 02-Apr-2009 Process based (Test Conditions) 02-Apr-2009 26-Jun-2009 Report 26-Jun-2009 This statementalso confirms thatthis final reportreflects the raw data. Quality Assurance: Ms. S. Zeugin

## _>.2o//~

............

# {,/'.
............................................ Date:

## ~~ 

# /JJ2oo,

Page6Harlan Laboratories Study C31177 Leuco SulfurBlack 1 

## PREFACE General Information Test Item: Study Title: Sponsor: StudyMonitor: Test Facility: QA:


### Responsibilities Study Director: Deputy StudyDirector: Head ofQA: 

### Schedule Experimental StartingDate: Experimental Completion Date: Report

Leuco SulfurBlack 1 Ready BiodegradabilityofLeuco SulfurBlack 1 in a Manometric Respirometry Test ClariantProducts (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. U. Memmert Dr. G. Eisner Mr. T. Fink

23-Mar-2009 23-Apr-2009

Page 7Harlan Laboratories Study C31177 Leuco Sulfur Black I Report


### Data Requirements I 

### Test Guidelines This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: • OECD Guideline for Testing of Chemicals No. 301 F, Ready Biodegradability: Manometric Respirometry Test, 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.4-D, Manometric Respirometry Test. • EU Commission Directive 92/69 EEC, C.4-D, Manometric Respirometry Test, 1992. 

### Summary ofStudy Plan Amendments First Amendment to Study Plan

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

### Archiving Harlan Laboratories Ltd. (4452 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 8Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## DEFINITIONS BOD

ThOD

COD Report

The biochemical oxygen demand (mg) is the amount of oxygen consumed by micro-organisms when metabolizing a test compound; also expressed as mg oxygen uptake per mg test compound. The theoretical oxygen demand (mg) is the total amount of oxygen required to oxidize a chemical completely; it is calculated from the molecular formula and is also expressed as mg oxygen required per mg test compound. The chemical oxygen demand (mg) is the amount of oxygen consumed during oxidation ofa test compound with hot, acidic dichromate; it provides a measure of the amount ofoxidizable matter present; also expressed as mg oxygen consumedpermg test compound.

Page 9Harlan Laboratories Study C31177 Leuco Sulfur Black I 

## 1 SUMMARY Report

The test item Leuco Sulfur Black 1 was investigated for its ready biodegradability in a manometric respirometry test over 28 days according to the EU Commission Directive 92/69 EEC, C.4-D Commission Regulation (EC) No 440/2008, C.4-D and the OECD Guideline for Testing ofChemicals No. 301 F. The biochemical oxygen demand (BOD) ofthe test item Leuco Sulfur Black 1 in the test media was in the normal range found for the inoculum controls. Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 28 days. In the toxicity control, containing both Leuco Sulfur Black 1 and the reference item sodium benzoate, Leuco Sulfur Black 1 had no inhibitory effect on the activity of activated sludge microorganisms at the tested concentration of 142 mg/L. In the procedure controls, the reference item sodium benzoate was degraded by an average of 80% by Exposure Day 14, and reached an average biodegradation of82% bythe end ofthe test (Day 28), thus confirming suitability ofthe activated sludge.

Page 10Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## 2 PURPOSE Report

The purpose ofthis study was to determine the ready biodegradability ofLeuco Sulfur Black 1in a biodegradation test by following the BOD (biochemical oxygen demand) ofthe test item under defined conditions by means ofmanometric methods over a period of28 days.

Page 11Harlan Laboratories Study C31177 Leuco Sulfur Black 1 Report 

## 3 MATERIALS AND METHODS 3.1 Test Item The test item and the following information were provided bythe Sponsor: Identity: CAS No.: Description: BatchNo.: Composition:

StabilityofTest Item: ExpiryDate: Storage Conditions: 

### 3.2 Reference Item Leuco Sulfur Black 1 66241-11-0 Thixotrophic, black, wet paste ESA0007595 Leuco Sulfur Black 1: 52.75% (calculatedby 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) Stableunder storage conditions 11-Jul-2011 Atroom temperature (range 20 ± 5°C), light protected.

The reference item and the following information were supplied byFluka (BioChemika): Identity: ProductNo.: Lot No.: Purity: Expiration Date (as given bythe supplier): Expiration Date (handled at Harlan Laboratories): Storage Conditions: Sodiumbenzoate 71295 1348422 99.8% Jun-2013 (date ofrecommended retest) 30-Jun-2013 (date ofrecommended retest) In tightly closed original container, at room temperature at about 20 °C.

Page 12Harlan Laboratories Study C31177 Leuco Sulfur Black I 

### 3.3 Test System Report

The test design and test system are consistent with the testing guidelines and should provide a rational basis to assess the readybiodegradability ofthe test itemunder aerobic conditions. The study was performed with aerobic activated sludge from a wastewater treatmentplant (ARA Ergolz II, Fiillinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap waterby centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot ofthe final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. Based on this ratio, calculated amounts of wet sludge were suspended in test water (see Section 3.4.1) to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period ofthree days prior to use, the sludge was aerated at room temperature. Prior to use, the sludge was first thoroughly mixed and then diluted with test water to a concentration of 1g per liter (dry weight basis). Based on the determined dry weight of this diluted activated sludge, defined amounts were added to test waterto obtain a final concentration of30 mg drymaterial per liter. 

### 3.4 Test Procedures 3.4.1 Test Water The test water was prepared according to the testing guidelines. Analytical grade salts were dissolved in purified waterto obtain the following stock solutions: 1) KRzP04 KzHP04 NazHP04 x 2H20 NH4Cl 8.50 giL 21.75 giL 33.40 giL 0.50 giL ThepH ofthis solution was 7.4. 2) 22.50 giL 3) CaClz x 2Hz0 36.40 giL 4) FeCh x 6Hz0 0.25 giL, stabilized with one drop ofconcentrated HCl perliter To obtain the final test water, 10 mL of stock solution No. 1 and 1 mL each of stock solution Nos. 2, 3 and 4 were combined and made up to 1000 mL with purified water. The pH was adjusted from 7.7 to 7.4 with a diluted hydrochloric acid solution.

Page l3Harlan Laboratories Study C31177 Leuco Sulfur Black 1 3.4.2 Test Concentrations Report

The test item was added to the designated test flasks with test water. No emulsifiers or solvents were used but ultrasound dispersion was employed for fifteen minutes to obtain a suspension of the test item as homogeneous as possible. The reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. A stock solution containing 2.5 g sodium benzoate per liter test water was prepared by completely dissolving 250 mg sodium benzoate in 100 mL test water. From this stock solution, 10 mL aliquots were added to the corresponding test flasks containing test water. Finally, activated sludge was added to each test flask (see Section 3.3). The final test volume was 250 mLpertest flask. Identification Replicate Amount oftest item1 Amount ofreference item1 No. mg!L COD2 mg!L ThOD3 Test item 1 140 111 

## --- n.a. Test item 2 141 112 

## --- n.a. Inoculum control 1 

## --- n.a. 

### --- n.a. Inoculum control 2 

## --- n.a. 

## --- n.a. Procedure control 1 

## --- n.a. 100 167 Procedure control 2 

### --- n.a. 100 167 Toxicity control4 1 142 113 100 167 The tabulated values represent rounded results obtained by calculationusing the exactraw data. Chemical oxygen demand in mg OiL test solution. Calculation ofthe ThOD was not possible due to the complex structure (composition) ofthe test item. The COD was not determined in compliance with the GLP regulations and is therefore excluded from the Statement ofCompliance in this report. However, the results will be archived under this study number. Theoretical oxygen demand in mg 0 2/L test solution. The test item can be assumed to be inhibitory ifin the toxicity control less than 25% degradation (based on total ThOD) occurs within 14 days. n.a. not applicable.

Page 14Harlan Laboratories Study C31177 Leuco SulfurBlack I Report


### 3.4.3 Test Conditions Apparatus:

Principle:

Test duration: Light conditions: Test temperature: pH: The test flasks (500 mL reaction vessels, labeled with the necessary information to ensure unmistakable identification) were incubated under continuous stirring in a SAPROMAT Dl2 (Voith GmbH, Heidenheim, Germany). Oxygen consumption was recorded manually by taking a daily reading at least on each working day. Electro-chemical analysis process: The biodegradation process consumes the dissolved oxygen in the test medium and generates C02• The C02 is adsorbed by soda lime, which results in a decrease ofthe total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal bymeans of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from a copper sulfate solution. 28 days Darkness 22 °C, maintained with a built-in thermostat and checked once per week. Prior to test start, the pH was measured in each test flask before the addition of the activated sludge inoculum (Table 3). At the end of incubation, the pH was measured again in each test flask.

Page 15Harlan Laboratories Study C31177 Leuco Sulfur Black I Report


### 3.5 Calculations The tabulated values represent rounded results obtained by calculation using the exactraw data. The percentagebiodegradation was calculated according to the following formula: %biodegradation = BOD (mg0 2/mgchemical) x1 OO% COD and/orThOD (mg0 2/mgchemical) where: BOD = Biochemical oxygen demand ofthe test or reference item

COD ThOD = (mg0 2 uptake/Lbytestand/orreferenceitem)- (mg0 2 uptake/Lbyinoculumcontrol) mgtest and/orreferenceitemperliter Chemical oxygen demand ofthe test item (since the ThOD ofthe test item could not be calculated; see Section 3.6) Theoretical oxygen demand ofthe reference item The theoretical oxygen demand is the total amount ofoxygen required to oxidize a chemical completely. It is calculated from the molecular formula assuming the turnover ofH into H20, C into COz, P into Pz05, Na into NazO, halogens into their corresponding hydrogen halides and N into NH4 and/or N03 according to EU Commission Directive 92/69 EEC C.4-D andISO 9408 (1999). 

### 3.6 Determination ofthe Chemical Oxygen Demand Due to the complex structure (composition) ofthe test item, its chemical oxygen demand (COD) was determined according to the EU Commission Directive 92/69/EEC, C.6 following DIN 38414-S9. The COD was determined at BMG Engineering AG, Ifangstrasse 11, 8952 Schlieren I Switzerland. The COD determinationwas not performed in compliance with the GLP regulations. Therefore, it is excluded from the Statement of Compliance in this report. However, it was performed in compliance with ISOIIEC 17025. Theresults are archivedunderthis studynumber.

Page 16Harlan Laboratories Study C31177 Leuco SulfurBlack 1 Report 

## 4 RESULTS AND DISCUSSION 4.1 Biodegradation ofthe Test Item Theresults are presented in Table 1and Table 2, and in Figure 1andFigure 2. The percent biodegradation of the test item was calculated based on the chemical oxygen demand (COD) of0.795 mg 02/mg test item. The biochemical oxygen demand (BOD) ofthe test item Leuco Sulfur Black 1 in the test media was in the normalrange found for the inoculum controls throughout the studyperiod of28 days. Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 28 days. 

### 4.2 Biodegradation ofthe Reference Item Theresults are presentedin Table 1and Table 2, and in Figure 1and Figure 2. The percent biodegradation ofthe reference item sodium benzoate was calculated based on the theoretical oxygen demand of 1.67 mg 02/mg (ThOD; see Sections 3.4.2 and 3.5). In the procedure controls, the reference item was degraded by an average of 80% by Exposure Day 14, thus confirming suitability ofthe activated sludge. At the end ofthe test (Day 28), the reference item was degradedbyan average of82%.

Page 17Harlan Laboratories Study C31177 Leuco SulfurBlack 1 Report


### 4.3 Biodegradation in the Toxicity Control The results arepresented in Table 1and Table 2, and in Figure 1and Figure 2. The percent biodegradation in the toxicity control, containingboth the test item and the reference item, was calculated based on the COD ofthe test item and the ThOD of the reference item (see Sections 3.4.2, 3.5 and 3.6). In

### the toxicity control, the course ofbiodegradation over the 28-day exposure period was similar to the two procedure controls, containing only the reference item. Within 14 days ofexposure, biodegradation amountedto 50%. Thus, according to the test guidelines, the test item had no inhibitory effect on activated sludge microorganisms at the tested concentration of 142 mg/L because biodegradation in the toxicity control was >25% within 14 days. 

### 4.4 Chemical Oxygen Demand (COD) ofthe Test Item The chemical oxygen demand (COD) of the test item Leuco Sulfur Black 1 was determined according to the EU Commission Directive 92/69/EEC, Part C.6 following DIN 38414-S9. The duplicate values were 0.78 and 0.81 mg 

### Oz/mg test item. The mean COD was 0.795 mg 

### Oz/mg test item. 

### 4.5 pH Measurement Theresults are presented in Table 3. The pH measured in all flasks at the start ofthe test was 7.4. At the end ofexposure (Day 28), pH values of7.1-7.8 were measured.

Page 18Harlan Laboratories Study C31177 Leuco SulfurBlack 1 

## 5 TABLES Report

The tabulatedvalues representroundedresults obtainedbycalculationusing the exactraw data. Table 1 Oxygen Consumption in the Test Flasks Cumulativeoxygen consumption (mg/L) Test item Inoculum Procedure Toxicity control control control Time Replicate No. Replicate No. Replicate No. Replicate No. (days) 1 2 1 2 1 2 1 0 0 0 0 0 0 0 0 1 0 0 0 0 32 35 40 2 

### -- -- 

### -- -- 

### -- -- -- 3
-- 

### -- -- -- -- -- -- 4 1 5 6 10 114 121 124 5 5 6 8 10 122 129 132 6 7 7 12 10 128 135 138 7 7 7 12 10 132 139 142 8 7 8 14 11 134 143 146 9 

### -- -- 

### -- -- 

### -- 

### -- -- 10 

### -- 

### -- -- -- 

### -- -- -- 11 9 9 20 12 144 150 154 12 9 10 24 13 146 153 156 13 9 10 24 13 148 154 158 14 9 10 26 14 152 156 160 15 

### -- -- -- 

### -- -- -- -- 16 

### -- -- -- 

### -- -- -- -- 17 

### -- -- -- -- -- -- -- 18 9 10 30 14 156 162 164 19 9 11 32 14 156 164 166 20 9 11 34 15 158 165 168 21 9 13 38 16 158 167 168 22 9 13 38 16 160 167 170 23 

### -- -- -- -- -- -- -- 24 

### -- -- -- -- -- -- -- 25 9 13 42 16 160 169 172 26 9 13 42 16 162 170 172 27 9 13 44 16 162 170 172 28 9 14 46 16 164 171 174 No reading taken

Page 19Harlan Laboratories Study C31177 Leuco Sulfur Black 1 Report
Table 2 Biodegradation inthe Test Flasks PercentageBiodegradation1 Testitem Procedurecontrol Time Replicate No. Replicate No. (days) 1 2 1 2 0 0 0 0 0 1 0 0 19 21 2 

### -- -- -- -- 3 

## -- -- -- 

## -- 4 -6 -3 63 68 5 -4 -3 68 72 6 -4 -4 70 74 7 -4 -4 72 77 8 -5 -4 73 78 9 

### -- -- -- -- 10 

### -- -- -- -- 11 -6 -6 77 80 12 -9 -8 76 81 13 -9 -8 78 81 14 -10 -9 79 81 15 

### -- -- -- -- 16 

### -- -- 

### -- -- 17 

### -- -- -- -- 18 -12 -11 80 84 19 -13 -11 80 84 20 -14 -12 80 84 21 -16 -12 78 84 22 -16 -12 80 84 23 

### -- -- -- 

### -- 24 

### -- -- -- -- 25 -18 -14 78 84 26 -18 -14 80 84 27 -19 -15 79 84 28 -20 -15 80 84 Mean (Day 28) (-17) 

### * 82 Corrected for the mean oxygen uptake ofthe inoculum controls (see Section 3.5)

-- Notdetermined Toxicity control Replicate No. 1 0 14



### --

-- 41 44 45 47 48



### --

-- 49 49 50 50



### --

--

-- 51 51 51 50 51



### --

-- 51 51 51 51 not applicable 

### * Negative value due to higher oxygen consumption in the inoculum controls than in the test flasks with test item. Conclusion: Biodegradation after 28 days

### =0 o/o

Page 20Harlan Laboratories Study C31177 Leuco Sulfur Black I Report
Table 3 pHValues at the Start and at the End ofthe Test

Replicate Identification pH No. Start

1 Test item 7.4 2 Test item 7.4 1 Inoculum control 7.4 2 Inoculum control 7.4 1 Procedure control 7.4 2 Procedure control 7.4 1 Toxicity control 7.4 End 7.1 7.1 7.1 7.1 7.8 7.8 7.7

Page 21Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## 6 

## FIGURES Report

Figure 1 Oxygen Consumption in the TestFlasks duringthe Incubation Period
200 180

## ~ 160 0 140 I>J) 

### !. 120 

## = Q ·..:: c. 100 

### e 

## = "' 80 

### = Q Col 

## = 60 

### ... I>J)
..... >'I 0 40 20

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. •11: •. : : . .•·• • · · · · · · ·• ·•·•: •.t:.. t ..**~.~.;**..;,.: .. t ..:t.:l,:.l**-~:**

### • i 

## ·I·il;..;,;...........& ............., ...,

..,,...........,,e:::l!!l....i!!i..·il!i................... j5.•i!!i..··i5···2!:**.,,.,. 11** **,1!:~:~:~ ·::~~·1""','.'1**  I I I I I I I I I I l I I I I I I I I I 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Exposure time (days) """"""'I>""""""'Test item

......................Test item
· · ·E>· · · Inoculum control
· · ·•·· · Inoculumcontrol --e-- Procedure control -----Procedurecontrol

- •>E ·-Toxicity control

Page 22Harlan Laboratories Study C31177 Leuco Sulfur Black I Report Figure 2 Biodegradation in the Test Flasks during the Incubation Period
100 80 60**~** ::!f.**~** 

### = 40 0**·~** 

# .. "0 

# .. ... t>ll 20 

# .. "0**-~ ~**  0 -20 -40**~** - .**-~-X~·**

## *·-·-·**~-** 

## *·*-x-"* - . -***""X~·** :K

....**~x** x·"

2 4 6 8 10

Exposure time (days) (based on values presented in Table 2) --+--Test item ---..-Test item ---e-- Procedure control --4- Procedure control**-·~·-Toxicity** control

Page 23Harlan Laboratories Study C31177 Leuco Sulfur Black 1 Report



## APPENDIX I- FINALIZED STUDYPLANAND FIRST AMENDMENTTOSTUDYPLAN

Page 24

## STUDY PLAN Leuco SulfurBlack 1 Ready BiodegradabilityofLeuco SulfurBlack 1 in a Manometric Respirometry Test

StudyDirector: Test Facility:
Sponsor:
StudyIdentification:
Version: Dr. Ulrich Memmert 

### Harlan Laboratories Ltd. Zelgliweg 1 4452 ltingen I Switzerland ClariantProducts (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1 I Switzerland Harlan Laboratories Study C31177
Final
Page 1of 16

# harlant~ 

# ...

Page 25Harlan Laboratories Study C31177 Leuco Sulfur Black l 

## CONTENTS Study Plan Page2



### CONTENTS ....................................................................................................................................2 SIGNATlJRES ................................................................................................................................ 3 PREFACE .......................................................................................................................................4 General Information...............................................................................................................4 Responsibilities......................................................................................................................4 Schedule.................................................................................................................................4 Good Laboratory Practice......................................................................................................5 Data Requirements I

### Test Guidelines ....................................................................................5 Amendment and Deviation Procedures .................................................................................5 Archiving ............................................................................................................................... 6 DEFINITIONS ................................................................................................................................7 1 PURPOSE..............................................................................................................................8 2 MATERIALS AND METHODS...........................................................................................9 2.1 Test Item ..................................................................................................................9 2.2 Reference Item.........................................................................................................9 2.3 Test System............................................................................................................ 10 2.4 Test Procedures...................................................................................................... 11 2.4.1 TestWater ............................................................................................... 11 2.4.2 Test Concentrations................................................................................. 12 2.4.3 Test Conditions........................................................................................ 13 2.4.4 Calculations............................................................................................. 14 2.4.5 Determination ofthe COD ...................................................................... 15 2.4.6 Determination ofNitrification................................................................. 15 3 CONDITIONS FORTHE VALIDITY OF THE TEST...................................................... 16 4 REPORTING....................................................................................................................... 16 5 DISTRIBUTION ................................................................................................................. 16

Page 26Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## SIGNATURES Study Director:

Management:

Quality Assurance:

For the Sponsor: Study Plan Page 3

Dr. Ulrich Memmert

Dr. JosefMagyar



### Gabriele Stremmel

Dr Uwe Ensenbach Clariant Products (Switzerland) Ltd.



# ...**t::?~**

# ....**~**

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

## ,4~/t1**/t'i** Page 27Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## PREFACE General Information TestItem: Study Title: Sponsor: Study Monitor: TestFacility: QA:


### Responsibilities StudyDirector: Deputy StudyDirector: HeadofQA: 

### Schedule Anticipated Experimental StartingDate: AnticipatedExperimental Completion Date: Anticipated Date ofDraftReport: Study Plan

Leuco Sulfur Black 1 Ready Biodegradability ofLeuco Sulfur Black 1 in a Manometric Respirometry Test Clariant Products (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1 I Switzerland DrUwe Ensenbach HarlanLaboratories Ltd. Zelgliweg 1 4452 Itingen I Switzerland HarlanLaboratories Ltd. Quality Assurance GLP Zelgliweg 1 4452 Itingen I Switzerland

Dr. Ulrich Memmert Dr. Gottfried Eisner Mr. Tobias Fink

Mar/ Apr 2009 Apr/May 2009 Jun/Jul 2009 Page4

Page 28Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

### Good Laboratory Practice Study Plan Page 5

This study will be performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted May 18th, 2005 [RS 813.112.1]. This Ordinance is based on the OECD Principles ofGood Laboratory Practice, as revised in 1997 and adopted November 26th, 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). Rationale for selection ofBMG Engineering AG for COD determination: The COD (Chemical Oxygen Demand) ofthe test item is not determined at Harlan Laboratories Ltd. This work will be subcontracted to BMG Engineering AG, 8952 Schlieren I Switzerland, a company certified by the Swiss Testing Service to perform the COD determination according to ISO/IEC 17025. As the COD determination is not performed under GLP, it will be excluded from the Statement ofCompliance. 

### Data Requirements I 

### Test Guidelines The study will follow the procedures indicated by the following internationally accepted guidelines and recommendations: • OECD Guideline for Testing of Chemicals No. 301 F, Ready Biodegradability: Manometric Respirometry Test, 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.4-D, Manometric Respirometry Test. • EU Commission Directive 92/69 EEC, CA-D, Manometric Respirometry Test, 1992. 

### Amendment and Deviation Procedures Amendments (planned changes) to the study plan will be issued and signed bythe 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, 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 bepromptly informed ofany relevant deviations from the study plan.

Page 29Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

### 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 30Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## DEFINITIONS BOD

ThOD

COD Study Plan Page 7

The biochemical oxygen demand (mg) is the amount of oxygen consumed by micro-organisms when metabolizing a test compound; also expressed as mg oxygen uptake per mg test compound. The theoretical oxygen demand (mg) is the total amount of oxygen required to oxidize a chemical completely; it is calculated from the molecular formula and is also expressed as mg oxygen required per mg test compound. The chemical oxygen demand (mg) is the amount of oxygen consumed during oxidation ofa test compound with hot, acidic dichromate; it provides a measure of the amount ofoxidizable matter present; also expressed as mg oxygen consumedpermg test compound.

Page 31Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

## 1 PURPOSE Study Plan Page 8

The purpose ofthis study is to determine the ready biodegradability ofLeuco Sulfur Black 1in a biodegradation test by following the BOD (biochemical oxygen demand) ofthe test item under defined conditionsbymeans ofmanometric methods over a period ofusually 28 days.

Page 32Harlan Laboratories Study C31177 Leuco Sulfur Black I Study Plan Page 9 

## 2 MATERIALSAND METHODS 2.1 Test Item The test item and the following information were provided bythe Sponsor: Identity: Batch no.: Purity: Expiration date: Formulation or composition: Aggregate state Iphysical form at room temperature: Color: Storage conditions: Safetyprecautions:


### 2.2 Reference Item Leuco Sulfur Black 1 ESA0007595, 11.07.08 45.5% 11-July-2011 Confidential information; available in Sponsor's file Paste ILiquid Black Atroom temperature at about 20 °C, away from direct sunlight. Routine hygienic procedures are sufficient to ensure personnel health and safety.

The reference item and the following information were supplied by Fluka (BioChemika): Identity: Product no.: Lot no.: Purity: Expiration date, as given bythe supplier: Expiration date, handled at Harlan Laboratories: Storage conditions: Safetyprecautions: Sodium benzoate 71295 1348422 99.8% Jun-2013 (date ofrecommended retest) 30-Jun-2013 (date ofrecommended retest) In tightly closed original container, at room temperature at about 20 °C. Routine hygienic procedures are sufficient to ensure personnel health and safety.

Page 33Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

### 2.3 Test System Study Plan Page 10

The test method and test system are consistent with the testing guidelines and should provide a rational basis to assess the ready biodegradability ofthe test item under aerobic conditions. The study will be performed with aerobic activated sludge from a local wastewater treatment plant, treating predominantly domestic wastewater. The sludge will be washed by centrifugation, the supernatant liquid phase will be decanted and the solid material resuspended in tap water. If necessary, this procedure will be repeated. A homogenized aliquot ofthe final sludge suspension will be weighed, thereafter dried and the ratio ofwet to dry weight will be calculated. Based on this ratio, calculated amounts of wet sludge will be suspended in test water (see Section 2.4.1) to obtain a concentration equivalent to 4 g (±10%) dry material per liter. During the holding period of maximum seven days prior to use, the sludge will be aerated at room temperature. Prior to use, the sludge will be diluted with test water to a concentration of 1 g dry material per liter. Defined volumes ofthis diluted activated sludge will be added to test water to obtain a final concentration of30 mg dry material perliter. Preparation and holding of the activated sludge may be performed within a different Harlan Laboratories study. However, the raw data or copies ofthe raw data ofthis work will be archived under this study number.

Page 34Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

### 2.4 Test Procedures 2.4.1 Test Water Study Plan Page 11

The test water will be prepared according to the testing guidelines. Analytical grade salts will be dissolved in purified water to obtain the following stock solutions: 1) KH2P04 8.50 giL K2HP04 21.75 giL Na2HP04 x 2H20 33.40 giL NH4Cl 0.50 giL The pH ofthis solution should be 7.4. 22.50 g/L 36.40 giL 0.25 giL, stabilized with one drop ofconcentrated HCl per liter To obtain the final test water, 10 mL of stock solution No. 1 and 1mL each of stock solution Nos. 2, 3 and 4 will be combined and made up to 1000 mL with purified water. Ifnecessary, the pH will be adjusted to 7.4 ± 0.2 with a diluted hydrochloric acid or sodium hydroxide solution.

Page 35Harlan Laboratories Study C31177 Leuco Sulfur Black I
2.4.2 Test Concentrations Study Plan Page 12

The test item will be directly added to the test flasks. No emulsifiers or solvents will beused, but ultrasound dispersion maybe employed to obtain a homogeneous suspension ofthe test item. The reference item sodium benzoate will be tested simultaneously under the same conditions as the test item, and will function as a procedure control. Sodium benzoate will be directly weighed into the test flasks or added via concentrated stock solution. To each flask activated sludge will be added (see Section 2.3). Finally, the flasks will be made up to a volume of250 mL with test water. Identification No. of Amount oftest item Amount ofreference item test mg/L 

## coo• (mg 02/L) mg/L ThOD1 flasks (mg 02/L) Testitem 2 about 100 min 50-100 

### --- n.a. Procedure control2 2 

## --- n.a. about 100 min 50-100 Inoculum control2 2 

### --- n.a. 

### --- n.a. Toxicity control 1 about 100 min 50-100 about 100 min 50-100 Chemical oxygen demand (COD) and theoretical oxygen demand (ThOD) in mg 0 2 /L test solution Control flasks may also beused for other Harlan Laboratories studies which may run in parallel. n.a.: not applicable

Page 36Harlan Laboratories Study C31177 Leuco Sulfur Black 1 Study Plan Page 13


### 2.4.3 Test Conditions Apparatus:

Principle:

Test duration: Light conditions: Test temperature: pH: The test flasks (500 mL reaction vessels, labeled with the necessary information to ensure unmistakable identification) will be incubated under continuous stirring in a SAPROMAT Dl2 (Voith GmbH, Heidenheim, Germany). Oxygen consumption will be recorded manually by taking a daily reading at least on each working day. Electro-chemical analysis process: The biodegradation process consumes the dissolved oxygen in the test medium and generates 

### C02. The 

### C02 is adsorbed by soda lime, which will result in a decrease ofthe total pressure in the airtight test flasks. The pressure drop is detected and converted into an electrical signal by means of an electrode type manometer. The consumed oxygen is replaced by electrolytically generated oxygen from copper sulfate solution. Normally 28 days. Sampling may be ended before 28 days, if the biodegradation curve ofthe test item, or reference item has reached a plateau for at least three determinations Darkness 22 ± 1°C, maintained with a built-in thermostat and checked once per week. Prior to test start, the pH will be measured in all test flasks before the addition ofthe activated sludge (inoculum), and, ifnecessary, adjusted to pH 7.4 ± 0.2 using a diluted hydrochloric acid or sodium hydroxide solution. At the end ofincubation, the pH will be measured again in all test flasks.

Page 37Harlan Laboratories Study C31177 Leuco Sulfur Black I 

### 2.4.4 Calculations Study Plan

The percentagebiodegradation will be calculated according to the following formula: Page 14

The percentage biodegradation ofthe reference item will be calculated according to the below given formula:

01 b" d d . BOD (mg 0/mgchemical) 10001 10 10 egra atwn

## = x 10 ThOD (mg0/mgchemical) The percentage biodegradation ofthe test item will be calculated according to the below given formula:

01 b" d d . BOD (mg0/mgchemical) 10001 10 10 egra atwn = x 10 COD (mg0/mgchemical) where: BOD Biochemical oxygen demand ofthe test orreference item

ThOD (mg0 2 uptake/Lbytestand/orreferenceitem)- (mg0 2 uptake/Lbyinoculumcontrol) mgtest and/orreferenceitemperliter Theoretical oxygen demand ofthe reference item. The theoretical oxygen demand is the total amount ofoxygen required to oxidize a chemical completely. It is calculated from the structural formula, assuming the turnover ofH into H20, C into C02, P into P20s, Na into Na20, halogens into their corresponding hydrogen halides and N into 

### NH4 and/or N03 according to EU Commission Directive 92/69 EEC C.4-D, Commission Regulation (EC) No 440/200 CA-D and ISO 9408 (1999). COD Chemical oxygen demand of the test item (if the ThOD cannot be calculated; see below)

Page 38Harlan Laboratories Study C31177 Leuco Sulfur Black I 

### 2.4.5 Determination ofthe COD Study Plan Page 15

The chemical oxygen demand (COD) of the test item will be determined according to the EU Commission Directive 92/69/EEC, Part C.6 following DIN 38414-S9 or DIN 38409-H41. The COD will be determined at BMG Engineering AG, Ifangstrasse 11, 8952 Schlieren 

### I Switzerland. The COD determination will not be performed in compliance with the GLP regulations and will therefore be excluded from the Statement of Compliance in the final report. However, BMG Engineering AG is certified by the Swiss Testing Service to perform the COD determination according to ISOIIEC 17025. The results will be archived under this study number. 

### 2.4.6 Determination ofNitrification If the test item contains nitrogen and in case of a significant biological oxygen demand, the concentrations ofnitrite and nitrate will be measured at least in the test media containing the test item and in the inoculum controls at the end ofthe test. The method used will be documented in the raw data and given in the report. According to the guidelines, the measured nitrite and nitrate concentrations will be used for the calculation ofthe biological oxygen demand caused bynitrification, ifnecessary.

Page 39Harlan Laboratories Study C31177 Leuco SulfurBlack 1 Study Plan 

## 3 CONDITIONS FOR THE VALIDITY OF THE TEST The results are considered valid ifthe following criteria are met: Page 16

- The oxygen demand of the inoculum control (medium and inoculum) is normally 20-30 mg 0 2 /L and should not be greater than 60 mg 02/L within 28 days.

- The degradation rates ofthe replicate test flasks containing the test item, test water and inoculum should not deviate by more than 20% at the time the plateau is reached, at the end ofthe test or at the end ofthe 10-daywindow.

- The percentage degradation of the reference item must reach the level for ready biodegradability (at least 60% ofThOD in a 10-daywindow) byDay 14. 

## 4 REPORTING A GLP-compliant draft report will be submitted to the Sponsor for scientific review. Following receipt ofthe Sponsor's comments, a QA-audited final report will be issued. 

## 5 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 40

## FIRSTAMENDMENT TO STUDY PLAN
Leuco SulfurBlack 1 Ready Biodegradability ofLeuco Sulfur Black 1 in a Manometric Respirometry Test
StudyDirector: TestFacility:
Sponsor:
StudyIdentification:
Version: Dr. U. Memmert 

### Harlan Laboratories Ltd. Zelgliweg 

### 1 4452 Itingen I Switzerland Clariant Products (Switzerland) Ltd. Rothausstrasse 61 4132 Muttenz 1 I Switzerland
Harlan Laboratories Study C31177
Final
Page 1 of5 

# harlanrr· 

# ...

Page 41Harlan Laboratories Study C31177 Leuco SulfurBlack I 

### SIGNATURES Study Director:

Forthe Sponsor: FirstAmendmentto Study Plan Page2



### Dr. U. Memmert



# ...**/P~** Date:

# ~

# Jf**Z~#/** DrU. Ensenbach Clariant Products (Switzerland) Ltd.



# ..**f.~**

# ....... Date: 

# ?dt 

# tr;**!t!~f**

Page 42Harlan Laboratories Study C31177 Leuco Sulfur Black I 

## PAGE 9 

## CONCERNING First Amendment to Study Plan Page 3



### 2 MATERIALS AND METHODS 2.1 Test Item 

## PRESENT Identity: Batchno.: Purity: Expiration date: Formulation orcomposition: Aggregate state Iphysical form at room temperature: Color: Storage conditions: Safetyprecautions: Leuco Sulfur Black 1 ESA0007595, 11.07.08 45.5% 11-July-2011 Confidential information; available in Sponsor's file Paste ILiquid Black Atroom temperature at about 20 °C, away from direct sunlight. Routine hygienic procedures are sufficient to ensure personnel health and safety.

Page 43Harlan Laboratories Study C31177 Leuco Sulfur Black 1 

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

Stability ofTest Item: Expiry Date: Storage Conditions: Safetyprecautions: First Amendment to 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: <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 Atroom temperature (range 20 ± 5°C), lightprotected. Routine hygienic procedures are sufficient to ensure personnel health and safety. 

## REASON FORTHEALTERATION Standardized test item informationrequested bythe Sponsor.

Page 44Harlan Laboratories Study C3ll77 Leuco Sulfur Black l 

## DISTRIBUTION First Amendment to Study Plan

This study plan amendment will be distributed as follows: Sponsor Sponsor: Harlan Laboratories Ltd. StudyFile: Study Director: Deputy Study Director: Technical Coordinator: Logistics: 

### QAESM:



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

Page 45

## CONTAINS 

## NO Ct