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 

Ready Biodegradability of Leuco Sulfur Black 1 

in a Manometric Respirometry Test 

Study Director: 

Test Facility: 

Sponsor: 

Dr. U. Memmert 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Study Identification: Harlan Laboratories Study C31177 

Version: Final 

Study Completion Date: 19-Aug-2009 

Page 1 of45 

CONTAtNS NO CBt 

haria nTh 

... 

\'\'-\~ 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 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 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 of the Chemical Oxygen Demand ................................................. 16 

4 RESULTS AND DISCUSSION .......................................................................................... 17 

4.1 Biodegradation of the Test Item ............................................................................ 17 

4.2 Biodegradation of the Reference Item ................................................................... 17 

4.3 Biodegradation in the Toxicity Control ................................................................. 18 

4.4 Chemical Oxygen Demand (COD) of the 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 

JAI I ~riD! l~i(};) .&.:}2l 

OOIT AM~C lVH ,,;;;. ::JV11,>i.:l 

J~OITAV1 Vl!Af\·K'O TC\~1 <:'ir:n (a•t Q 3t W.H r 4.~.~\--l', -tj..A• ·,. ',:;~,_:·n;:• 

Page 2 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

LIST OF TABLES 

Report 

Table 1 Oxygen Consumption in the Test Flasks .................................................................... 19 

Table 2 Biodegradation in the Test Flasks ............................................................................... 20 

Table 3 pH Values at the Start and at the End of the Test.. ..................................................... 21 

LIST OF FIGURES 

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 3 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

GLP CERTIFICATE 

Schweizerische Eidgenossensch~ft 

Conf~d~ration suisse 

Confederazione Svizzera 

Conflderniun svizra 

Swiss Confederation 

Report 

The Swin GlP Monlkrtlg Authortlt• 

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 for the: !nvlronment PO!N 

/ sw1ssmedic 

Statement of GLP 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 

of the test facility: 

Areas of expertise according to 

article 3 paragraph 1 letter d OGLP: 

Har1an Laboratories Ltd. 

Zelgliweg 1 

4452 Wngen, Switzer1and. 

1 J Physical-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) I Swiss Agency for Therapeutic Products (Swissmedic) 

Date of inspection: 05th to 09th and 26th to 3oth November 2007 

Date of decision: 30th April 2008 

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

is able to conduct studies according to the 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 

llwnotilcetlcn auhlrityfOrehemlella Is fie COOI'tiiM!iOn and dadllon ~for lhl good llboralory prdc. (GLPI far the FOEN, lhe FOPH and 8wl:umec:llc. 

tMtu Fad'.., O!nDe cA Pt.tlllc Health, Con1uMt pn;Mttion d.-.ct«ate, No!lllcallon IWihorilyforc;:temicala, CH-3003 Sem. 

""'*EmDen Phone: +41l0)313227305 Fax: +41 (0)3132354 8S 

Page4 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

GOOD LADORA TORY PRACTICE 

Report 

STATEMENT OF COMPLIANCE 

Harlan Laboratories Study: 

Test Item: 

Study Director: 

C31177 

Leuco Sulfur Black I 

Dr. U. Memmert 

Study Title: Ready Biodegradability of Leuco Sulfur Black 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 of Good Laboratory Practice, as revised in 1997 and adopted November 261

\ 

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

These principles are compatible with Good Laboratory Practice regulations specified by 

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

and Japan (MHLW, MAFF and MET!). 

Exclusions: 

Solubility pre-experiment of the test item in test water. 

Determination of the COD of the test item. 

Rationale for selection of BMG 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 of Compliance. 

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

Study Director: Dr. U. Memmert 

...... !!M:Jt. .................... . Date:~~~ 

Page 5 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

Report 

QUALITY ASSURANCE GLP STATEMENT 

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: Ready Biodegradability of Leuco Sulfur Black 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 and the 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 of Reports 

Dates and Types of QA Inspections to the Study Director 

and to the Test Facility 

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

Quality Assurance: Ms. S. Zeugin 

_>.2o//~ 

............ {,/'. ........................................... . 

Date: ~~ /JJ 2oo, 

Page6 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Head ofQA: 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Report 

Leuco Sulfur Black 1 

Ready Biodegradability of Leuco Sulfur Black 1 in a 

Manometric Respirometry 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. U. Memmert 

Dr. G. Eisner 

Mr. T. Fink 

23-Mar-2009 

23-Apr-2009 

Page 7 
Harlan 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 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.4-D, Manometric Respirometry Test. 

• EU Commission Directive 92/69 EEC, C.4-D, Manometric Respirometry Test, 1992. 

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 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 8 
Harlan 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 of a test compound 

with hot, acidic dichromate; it provides a measure of 

the amount of oxidizable matter present; also expressed 

as mg oxygen consumed per mg test compound. 

Page 9 
Harlan 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 of Chemicals No. 301 F. 

The biochemical oxygen demand (BOD) of the 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 of 82% by the end of the test 

(Day 28), thus confirming suitability of the activated sludge. 

Page 10 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

2 PURPOSE 

Report 

The purpose of this study was to determine the ready biodegradability of Leuco Sulfur Black 1 in 

a biodegradation test by following the BOD (biochemical oxygen demand) of the test item under 

defined conditions by means of manometric methods over a period of 28 days. 

Page 11 
Harlan 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 by the Sponsor: 

Identity: 

CAS No.: 

Description: 

Batch No.: 

Composition: 

Stability of Test Item: 

Expiry Date: 

Storage Conditions: 

3.2 Reference Item 

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 reference item and the following information were supplied by Fluka (BioChemika): 

Identity: 

Product No.: 

Lot No.: 

Purity: 

Expiration Date 

(as given by the supplier): 

Expiration Date 

(handled at Harlan Laboratories): 

Storage Conditions: 

Sodium benzoate 

71295 

1348422 

99.8% 

Jun-2013 (date of recommended retest) 

30-Jun-2013 (date of recommended retest) 

In tightly closed original container, at room temperature 

at about 20 °C. 

Page 12 
Harlan 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 ready biodegradability of the test item under aerobic conditions. 

The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA 

Ergolz II, Fiillinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was 

washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A 

homogenized aliquot of the 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 of three 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 1 g per liter (dry weight basis). Based on the determined dry weight of this 

diluted activated sludge, defined amounts were added to test water to obtain a final concentration 

of 30 mg dry material 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 water to obtain the following stock solutions: 

1) KRzP04 

KzHP04 

NazHP04 x 2H20 

NH4Cl 

8.50 giL 

21.75 giL 

33.40 giL 

0.50 giL 

The pH of this 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 of concentrated HCl per liter 

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 l3 
Harlan 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 mL per test flask. 

Identification Replicate Amount of test item 1 Amount of reference 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 calculation using the exact raw data. 

Chemical oxygen demand in mg OiL test solution. Calculation of the ThOD was not possible due to the 

complex structure (composition) of the test item. The COD was not determined in compliance with the GLP 

regulations and is therefore excluded from the Statement of Compliance 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 if in the toxicity control less than 25% degradation (based on 

total ThOD) occurs within 14 days. 

n.a. not applicable. 

Page 14 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 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 of the 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 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 15 
Harlan Laboratories Study C31177 

Leuco Sulfur Black I 

Report 

3.5 Calculations 

The tabulated values represent rounded results obtained by calculation using the exact raw data. 

The percentage biodegradation was calculated according to the following formula: 

% biodegradation = BOD (mg 0 2/mg chemical) x 1 OO% 

COD and/or ThOD (mg 0 2/mg chemical) 

where: 

BOD = Biochemical oxygen demand of the test or reference item 

COD 

ThOD 

= ( mg 0 2 uptake/L by test and/or reference item)- (mg 0 2 uptake/L by inoculum control) 

mg test and/or reference item per liter 

Chemical oxygen demand of the test item (since the ThOD of the test item could not 

be calculated; see Section 3.6) 

Theoretical oxygen demand of the reference item 

The theoretical oxygen demand is the total amount of oxygen required to oxidize a 

chemical completely. It is calculated from the molecular formula assuming the 

turnover of H 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 and ISO 9408 (1999). 

3.6 Determination of the Chemical Oxygen Demand 

Due to the complex structure (composition) of the 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 determination was 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. The results are archived under this study number. 

Page 16 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

Report 

4 RESULTS AND DISCUSSION 

4.1 Biodegradation of the Test Item 

The results are presented in Table 1 and Table 2, and in Figure 1 and Figure 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) of the test item Leuco Sulfur Black 1 in the test media 

was in the normal range found for the inoculum controls throughout the study period of 28 days. 

Consequently, Leuco Sulfur Black 1 was not biodegradable under the test conditions within 

28 days. 

4.2 Biodegradation of the Reference Item 

The results are presented in Table 1 and Table 2, and in Figure 1 and Figure 2. 

The percent biodegradation of the 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 of the activated sludge. At the end of the test (Day 28), the 

reference item was degraded by an average of 82%. 

Page 17 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

Report 

4.3 Biodegradation in the Toxicity Control 

The results are presented in Table 1 and Table 2, and in Figure 1 and Figure 2. 

The percent biodegradation in the toxicity control, containing both the test item and the reference 

item, was calculated based on the COD of the 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 of biodegradation over the 28-day exposure period was similar 

to the two procedure controls, containing only the reference item. Within 14 days of exposure, 

biodegradation amounted to 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) of the 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 

The results are presented in Table 3. 

The pH measured in all flasks at the start of the test was 7 .4. At the end of exposure (Day 28), 

pH values of7.1-7.8 were measured. 

Page 18 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

5 TABLES 

Report 

The tabulated values represent rounded results obtained by calculation using the exact raw data. 

Table 1 Oxygen Consumption in the Test Flasks 

Cumulative oxygen 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 19 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

Report 

Table 2 Biodegradation in the Test Flasks 

Percentage Biodegradation 1 

Test item Procedure control 

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 of the inoculum controls (see Section 3.5) 

-- Not determined 

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 20 
Harlan Laboratories Study C31177 

Leuco Sulfur Black I 

Report 

Table 3 pH Values at the Start and at the End of the 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 21 
Harlan Laboratories Study C311 77 

Leuco Sulfur Black 1 

6 FIGURES 

Report 

Figure 1 Oxygen Consumption in the Test Flasks during the Incubation Period 

200 

180 

~ 160 

0 140 I>J) 

!. 120 = Q 

·..:: 

c. 100 e 

= 

= "' 80 Q 

Col 

= 60 ... 

I>J) 

..... 

>'I 

0 40 

20 

0 

.a·e· .0-o.o······-.o 

.a·E> 

. • 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 

· · ·•· · · Inoculum control 

--e--Procedure control 

-----Procedure control 

- • >E ·-Toxicity control 

Page 22 
Harlan Laboratories Study C31177 

Leuco Sulfur Black I 

Report 

Figure 2 Biodegradation in the Test Flasks during the Incubation Period 

100 

80 

60 ~ 

::!f. 

~ 

= 0 40 ·~ 

.. 

"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 23 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

Report 

APPENDIX I- FINALIZED STUDY PLAN AND FIRST 

AMENDMENTTOSTUDYPLAN 

Page 24 
STUDY PLAN 

Leuco Sulfur Black 1 

Ready Biodegradability of Leuco Sulfur Black 1 

in a Manometric Respirometry Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

Version: 

Dr. Ulrich Memmert 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 ltingen I Switzerland 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Harlan Laboratories Study C31177 

Final 

Page 1 of 16 

harlant~ ... 

Page 25 
Harlan 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 of the COD ...................................................................... 15 

2.4.6 Determination ofNitrification ................................................................. 15 

3 CONDITIONS FOR THE VALIDITY OF THE TEST ...................................................... 16 

4 REPORTING ....................................................................................................................... 16 

5 DISTRIBUTION ................................................................................................................. 16 

Page 26 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

SIGNATURES 

Study Director: 

Management: 

Quality Assurance: 

For the Sponsor: 

Study Plan Page 3 

Dr. Ulrich Memmert 

Dr. Josef Magyar 

Gabriele Stremmel 

Dr Uwe Ensenbach 

Clariant Products (Switzerland) Ltd . 

... t::?~ .... ~ ....................... . Date: ,4~/t1 /t'i 

Page 27 
Harlan Laboratories Study C31177 

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 

Leuco Sulfur Black 1 

Ready Biodegradability of Leuco Sulfur Black 1 

in a Manometric Respirometry Test 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 I Switzerland 

Dr Uwe Ensenbach 

Harlan Laboratories Ltd. 

Zelgliweg 1 

4452 Itingen I Switzerland 

Harlan Laboratories 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 28 
Harlan 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 of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 

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). 

Rationale for selection of BMG Engineering AG for COD determination: 

The COD (Chemical Oxygen Demand) of the 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 of Compliance. 

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 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.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 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, 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. 

Page 29 
Harlan 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 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 30 
Harlan 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 of a test compound 

with hot, acidic dichromate; it provides a measure of 

the amount of oxidizable matter present; also expressed 

as mg oxygen consumed per mg test compound. 

Page 31 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

1 PURPOSE 

Study Plan Page 8 

The purpose of this study is to determine the ready biodegradability of Leuco Sulfur Black 1 in a 

biodegradation test by following the BOD (biochemical oxygen demand) of the test item under 

defined conditions by means of manometric methods over a period of usually 28 days. 

Page 32 
Harlan Laboratories Study C31177 

Leuco Sulfur Black I 

Study Plan Page 9 

2 MATERIALS AND METHODS 

2.1 Test Item 

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

Identity: 

Batch no.: 

Purity: 

Expiration date: 

Formulation or composition: 

Aggregate state I physical form at 

room temperature: 

Color: 

Storage conditions: 

Safety precautions: 

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 I Liquid 

Black 

At room 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 by the 

supplier: 

Expiration date, handled at Harlan 

Laboratories: 

Storage conditions: 

Safety precautions: 

Sodium benzoate 

71295 

1348422 

99.8% 

Jun-2013 (date of recommended retest) 

30-Jun-2013 (date of recommended 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 33 
Harlan 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 of the 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 of the final sludge suspension 

will be weighed, thereafter dried and the ratio of wet 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 of this diluted activated sludge will be added to test water to 

obtain a final concentration of 30 mg dry material per liter. 

Preparation and holding of the activated sludge may be performed within a different Harlan 

Laboratories study. However, the raw data or copies of the raw data of this work will be archived 

under this study number. 

Page 34 
Harlan 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 of this solution should be 7.4. 

22.50 g/L 

36.40 giL 

0.25 giL, stabilized with one drop of concentrated HCl per liter 

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 will be combined and made up to 1000 mL with purified water. If necessary, the 

pH will be adjusted to 7.4 ± 0.2 with a diluted hydrochloric acid or sodium hydroxide solution. 

Page 35 
Harlan 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 be used, but 

ultrasound dispersion may be employed to obtain a homogeneous suspension of the 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 of 250 mL with test water. 

Identification No. of Amount of test item Amount of reference item 

test mg/L coo• (mg 02/L) mg/L ThOD1 

flasks (mg 02/L) 

Test item 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 be used for other Harlan Laboratories studies which may run in parallel. 

n.a.: not applicable 

Page 36 
Harlan 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 of the 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 of the 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 of the activated sludge (inoculum), and, if necessary, adjusted 

to pH 7.4 ± 0.2 using a diluted hydrochloric acid or sodium hydroxide 

solution. At the end of incubation, the pH will be measured again in all 

test flasks. 

Page 37 
Harlan Laboratories Study C31177 

Leuco Sulfur Black I 

2.4.4 Calculations 

Study Plan 

The percentage biodegradation will be calculated according to the following formula: 

Page 14 

The percentage biodegradation of the reference item will be calculated according to the below 

given formula: 

01 b" d d . BOD (mg 0/mg chemical) 10 10 egra atwn = x 10001 10 

ThOD (mg 0/mg chemical) 

The percentage biodegradation of the test item will be calculated according to the below given 

formula: 

01 b" d d . BOD (mg 0/mg chemical) 10 10 egra atwn = x 10001 10 

COD (mg 0/mg chemical) 

where: 

BOD Biochemical oxygen demand of the test or reference item 

ThOD 

(mg 0 2 uptake/L by test and/or reference item)- (mg 0 2 uptake/L by inoculum control) 

mg test and/or reference item per liter 

Theoretical oxygen demand of the reference item. 

The theoretical oxygen demand is the total amount of oxygen required to oxidize a 

chemical completely. It is calculated from the structural formula, assuming the 

turnover of H 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 38 
Harlan Laboratories Study C31177 

Leuco Sulfur Black I 

2.4.5 Determination of the 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 of Nitrification 

If the test item contains nitrogen and in case of a significant biological oxygen demand, the 

concentrations of nitrite and nitrate will be measured at least in the test media containing the test 

item and in the inoculum controls at the end of the 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 of the biological oxygen demand caused by nitrification, if necessary. 

Page 39 
Harlan Laboratories Study C31177 

Leuco Sulfur Black 1 

Study Plan 

3 CONDITIONS FOR THE VALIDITY OF THE TEST 

The results are considered valid if the 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 of the 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 of the test or at the end ofthe 10-day window. 

- The percentage degradation of the reference item must reach the level for ready 

biodegradability (at least 60% ofThOD in a 10-day window) by Day 14. 

4 REPORTING 

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. 

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 
FIRST AMENDMENT TO STUDY PLAN 

Leuco Sulfur Black 1 

Ready Biodegradability of Leuco Sulfur Black 1 

in a Manometric Respirometry Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Identification: 

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 of 5 

harlanrr· 

... 

Page 41 
Harlan Laboratories Study C31177 

Leuco Sulfur Black I 

SIGNATURES 

Study Director: 

For the Sponsor: 

First Amendment to Study Plan Page2 

Dr. U. Memmert 

Date: 

... /P~ ~ Jf Z~#/ 

Dr U. Ensenbach 

Clariant Products (Switzerland) Ltd . 

.. f.~ ....... 

Date: ?dt tr; !t!~f 

Page 42 
Harlan 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: 

Batch no.: 

Purity: 

Expiration date: 

Formulation or composition: 

Aggregate state I physical form at 

room temperature: 

Color: 

Storage conditions: 

Safety precautions: 

Leuco Sulfur Black 1 

ESA0007595, 11.07.08 

45.5% 

11-July-2011 

Confidential information; available in Sponsor's file 

Paste I Liquid 

Black 

At room temperature at about 20 °C, away from direct 

sunlight. 

Routine hygienic procedures are sufficient to ensure 

personnel health and safety. 

Page 43 
Harlan Laboratories Study C31177 

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-20 11 

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. 

Page 44 
Harlan 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. 

Study File: 

Study Director: 

Deputy Study Director: 

Technical Coordinator: 

Logistics: 

QAESM: 

... 

1 x copy 

1 x (original) 

1 x (copy) 

1 x (copy) 

1 x (copy) 

1 x (copy) 

1 x (copy) 

Page 5 

Page 45 
CONTAINS NO Ct