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### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D

.C., 20460



### VIA FACSIMILE: 201-437-4801 April 19,2012 Attention: Glenn Merritt (201-437-4000) OFFICE OF PREVE NTION, PESTICIDESAND TOXIC SUBSTANCES



### Subject: Follow-up to fax transmission of4/13/12 (ePMA

### removal ofCBI claimsregarding identified studies) 

## DearMr. Merritt: This is sent to you pursuant ourconversation ofseveral minutes ago. In Larry Robinson's fax of April 13th 

### while there is a waiver ofconfidential business information (CBI) claims in the identified studies there is a an assertion ofcopyrightprotections and some additional text that provides in part
... "Reproduction, reference, summarization, retransmission or otheruse ofthis copyrighted document.

..including electronic transmission, using the internet oremail

....is strictly prohibited

...." While we understand the basis for the copyrightprotections, we have a concern that the language in the fax might be interpreted to mean that ePMA is asserting that the studies submitted perthe TSCA activity are not to bemade available in an Agencye-docket (electronic docket) orbe included in EPA websites among the collection ofTSCAhealth and safety studies. We are thinking that the identified limitations noted in the fax are not intended to apply to EPA's normal procedures for the handling ofnon-CBI materials submitted by entities subject to TSCA regulations (including the posting ofsuch materials ongovernment websites) and submittedby such entities in order to comply with a TSCA requirement, but are rather intended to limit entities outside the government from the unauthorized use or appropriation ofthe studies. Canyou please confirm CPMA's intent? Many thanks.

Scott M. Sherlock, Attorney Advisor Environmental Assistance Division Office ofPollution Prevention and Toxics

### CONFIDENTIAL BUSINESS INFORMATION



## REPORT

Study Title 

## ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH C.I. PIGMENT BLUE 61 (STATIC)
Author Ir.L.M. Bouwman Study completion date

23 October 2008

Test Facility NOTOX B.V. Hambakenwetering 7 5231 DD's-Hertogenbosch The Netherlands Laboratory Proiect Identification NOTOX Project 488233 NOTOX Substance 1836911A

-Page 1of22-C.I. Pigment Blue 61 NOTOX Project 488233 1. CONTENTS 1. CONTENTS...........................2 2. STATEMENT OF GLP COMPLIANCE .......................................................... 3 3. QUALITY ASSURANCE STATEMENT.......................................................... 4 4. SUMMARY...........................5 5. INTRODUCTION.................................................. 6 5.

1. Preface...........................6 5.2. Aim ofthe study.............................................................................. 6 5.3. Guidelines......................................................... 6 5.4. Storage and retention of records and materials............................................ 6 5.5. Definitions................................................................................... 7 6. MATERIALS AND METHODS.................................................................... 7 6.1. Test Substance............................................................................... 7 6.1.1. Test substance information........................................................... 7 6.1.2. Study specifictest substance information........................................... 7 6.1.3. Reference substance................................................................. 7 6.1.4. Preliminary data....................................................................... 8 6.2. Test system.................................................................................. 8 6.3. Breeding..................................................................................... 8 6.4. Preparation oftest solutions................................................................. 9 6.5. Limittest..................................................................................... 9 6.5.1. Test concentrations................................................................... 9 6.5.2. Test procedure and-conditions....................................................... 9 6.5.3. Sampling for analysis oftest concentrations ...................................... 10 6.5.4. Measurements and recordings ..................................................... 10 6.6. Electronic data capture..................................................................... 10 6.7. Interpretation ................................................................................ 11 6.7.1. Data handling ........................................................................ 11 6.7.2. Acceptability of the test.............................................................. 11 6.8. List ofdeviations ............................................................................ 11 6.8.1. Listof protocol deviations........................................................... 11 6.8.2. Listof standard operating procedures deviations ................................. 11 7. RESULTS....................................................................................... 11 7.

1. Measured concentrations................................................................... 11 7.2. Immobility.................................................................................. 11 7.3. Determination of effect concentrations.................................................... 12 7.4. Experimental conditions .................................................................... 12 8. CONCLUSION................................................................................... 12 TABLES Table 1 Acute immobilisation of daphnids after 24 and 48 hours ........................ 12 Table 2 Effect parameters.................................................................... 12 Table 3 pH and oxygen concentrations ..................................................... 12 APPENDICES APPENDIX I REFERENCE TEST................................................................. 13 APPENDIX 11 ANALYTICAL REPORT

-Page 2

-Cl. Pigment Blue 61 NOTOX Project488233 2. STATEMENT OF GLP COMPLIANCE NOTOX B..'s-Hertogenbosch, The Netherlands The study described inthis report has been correctly reported and was conducted incompliance with: The Organization for Economic Cooperation and Development (OECD) Good Laboratory Practice Guidelines (1997). Which essentially conform to: The United States Food and Drug Administration Good Laboratory Practice Regulations. The United States Environmental Protection Agency Good Laboratory Practice Regulations. The sponsor isresponsible for Good Laboratory Practice (GLP) compliance forall test substance information unless determined by NOTOX.

NOTOX B.V.
Ir. L.M. Bouwman Ing. E.J. van deWaart, M.Sc. Study Director Head of InVitro &Environmental Toxicology



### Date:.%$. 

## .... oO 

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

### ... £-9 CA60............

-Page 3

-C.1.Pigment Blue 61 NOTOX Project 488233 3. QUALITY ASSURANCE STATEMENT NOTOX B.V., 's-l-ertogenbosch, The Netherlands This report was inspected by the NOTOX QualityAssurance Unitto confirm that the methods and results accuratelyand completely reflect the raw data. The dates of Quality Assurance inspections are given below. During the on-site process inspections procedures applicable to this type of study were inspected. The reporting date isthe date of reporting to the Study Director. The QAU reportwas then forwarded tothe Test Facility Management.

Type of haerossStart End Reotn inpeton hae/rcesInspection Inspection daeot inspetionsdate date dt Study Protocol 09-Sep-08 09-Sep-08 09-Sep-08 Report 01-Oct-08 01-Oct-08 01-Oct-08 Process Environmental toxicology 14-Jul-08 18-Jul-08 18-Jul-08 Test substance handling Exposure Observations/Measurements Analytical and physical chemistry 25-Aug-08 25-Aug-08 01-Sep-08 Test substance handling Observations/Measurements

Head of Quality Assurance C.J.Mitchell B.Sc.

Date ... V .7.

-Page 4

-C.
. Pigment Blue 61 NOTOX Project488233 4. SUMMARY Acute Toxicity Study inDaphnia magna with C.l. Pigment Blue 61. The study procedures described inthis report were based on the OECD guideline No. 202, 2004. Inaddition, the procedures were designed to meet the test methods of the EEC directive 92/69, Part C.2, 1992, the ISO International Standard 6341, 1996 and the OECD series on testing and assessment number 23, 2000. The batch of C.1. Pigment Blue 61 tested was adark blue powder with granuleswith a purity of 96.830% and not completely soluble intest medium atthe loading rate prepared. Preparation of test solutions started with aloading rate of 100 mg/I applying a 10-minute treatment period with ultrasonic waves followed by a 3days of magnetic stirring to reach maximum solubility of the test substance inthe test medium. Subsequently, the aqueous mixture was filtered through amembrane filter (0.45 pm). The resulting filtrate was observed to be clear and light blue. Alimit testwas performed, exposing twenty daphnids per concentration (four replicates, five daphnids each) to the filtered solution prepared at aloading rate of 100 mg/I and acontrol group. The total test period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at thestart and the end ofthe test. Analysis of the samples taken atthe start of the test showed ameasured concentration of 57.8 pg/I at the loading rate of 100 mg/I. During the exposure period the measured concentration decreased to 40.2 pg/I (i.e. 69% of initial). Based on these results, the average exposure concentration was calculated to be 48 pg/I. The study met the acceptability criteria prescribed bythe protocol and was considered valid. 0.1. Pigment Blue 61 did not induce acute immobilisation of Daphnia magna at48 pg/I after 48 hours of exposure (NOEC). The 48h-EC 50 was >48 pg/I based on average exposure concentrations. This concentration was above the given water solubility limit of C.I. Pigment Blue 61 (i.e. 2.5 pg/I).

-Page 5

-C.I. Pigment Blue 61 NOTOX Project488233 5. INTRODUCTION 5.1. Preface Sponsor Color Pigments Manufacturers Association, Inc. 300 N.Washington St.Ste. 102, ALEXANDRIA, VA 22314 USA Study Monitor Mr. L.Robinson Test Facility NOTOX B. Hambakenwetering 7 5231 DD 's-Hertogenbosch The Netherlands Study Director Ir.L.M. Bouwman Technical Coordinator Ing. 'I.E. Carolus Principal Scientist Ir.M.J. C.Brekelmans Study Plan Start :15 September 2008 Completion 17 September 2008 5.2. Aim ofthe study The purpose of the study was to evaluate the test substancefor its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC 5oat24 and 48 hours ofexposure. 5.3. Guidelines The study procedures described inthis reportwere based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines forTesting of Chemicals, guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004. Inaddition, the procedures were designed to meet the test methods of the following guidelines and guidance documents: * European Economic Community (EEC), EEC directive 92169, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2. "Acute Toxicity for Daphnia". " ISO Intemnational Standard 6341: 'Water quality

- Determination of the inhibition ofthe mobility ofDaphnia magna Straus

- Acute toxicitytest, Third edition, 1998-04-01. * Guidance document on aquatic toxicitytesting of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000. 5.4. Storage and retention of records and materials Records and materials pertaining to the study including protocol, raw data, specimens (except specimens requiring refrigeration or freezing) and the final report are retained inthe NOTOX archives for a period ofat least 2years after finalization ofthe report. Afterthis period, the sponsorwill be contacted todetermine how the records and materials should be handled. NOTOX will retain information concerning decisions made.

-Page 6

-C.l1. Pigment Blue 61 NOTOX Project 488233 Those specimens requiring refrigeration or freezing will be retained by NOTOX for as long as the qualityof the specimens permits evaluation but no longer than three months afterfinalization of the report. NOTOX will retain atest substance sample until the expiry date, but no longer than 10 years after finalization ofthe report. Afterthis period the sample will be destroyed. 5.5. Definitions immobile are those animals not able to swim within 15 seconds after gentle agitation of the test vessel. The ECso isthe concentration oftest substance estimated to immobilise 50% of the daphnids after adefined period of exposure. No Observed Effect Concentration (NOEC) isthe highestconcentration tested at which no effect (i.e. immobilisation) is recorded.
6. MATERIALS AND METHODS 6.1. Test Substance 6.1.1. Testsubstance information Identification C.l. Pigment Blue 61 Structure

N

OH~ molecular formula C,,H29 N,0 3S Molecular weight 596 CAS Number 1324-76-1 Description Dark blue powderwith granules Batch 2084701 Purity 

### 96.830% Test substance storage At room temperature inthe dark stability under storage conditions Stable Expiry date 02 January 2018 6.1.2. Study specific test substance information Stability inwater At least96 hours Solubility inwater No 6.1.3. Reference substance This report includes the results of the most recent reference test with potassium dichromate (Appendix 1).

-Page 7

-C.
. Pigment Blue 61 NOTOX Project 488233 6.1.4. Preliminary data The solubility of Pigment Blue 61 inwater (bi dest.) at ambient temperature is2.5 pg/I (Clariant, SU-Technology &Innovation Management, Pigment Analytics). 6.2. Test system Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at leastthird generation, obtained by acyclical parthenogenesis under specified breeding conditions. Source In-house laboratory culture with aknown history. Reason for selection This system has been selected as an internationally accepted invertebrate species. Validity of batch Daphnids originated from ahealthy stock, 2nd to 5th brood, showing no signsof stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was nodelay 

### inthe production of the first brood. Characteristics For the testselection of young daphnidswith an age of < 24 hours, from parental daphnids of more than two weeks old. 6.3. Breeding Start of each batch With newborn daphnids, i.e. less than 3days old, by placing about 250 ofthem into 5 litres of medium inan all-glass culture vessel. Maximum age of the cultures 4weeks Renewal of the cultures After 7 days ofcultivation halfof the medium twice a week. Temperature of medium 18-22'C Feeding Daily, asuspension offresh water algae. Medium M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency 

### in Crustacea. An ultrastructural approach to antennal damage inDaphnia magna Straus. Protoplasma 154, 25-33). Composition of medium M7: Adjusted ISO medium: the following chemicals (analytical grade) are dissolved intap water purified by Reverse osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): Macrosalts: CaCI2.2H- 20 211.5 mg/I MgSO4.7H20 88.8 mg/I NaHCO 3 46.7 mg/I KCI 4.2 mg/I

-Page 8
-0.A. Pigment Blue 61 NOTOX Project 488233
Medium M7: trace elements, macro nutrients and vitamins are added tofreshly prepared ISO medium to reach the following concentrations: Trace elements: B 0.125 mg/I Fe 0.05 mg/I Mn 0.025 mg/I Li, Rb and Sr 0.0125 mg/I Mo 0.0063 mg/I Br 0.0025 mg/I cu 0.0016 mg/I Zn 0.0063 mg/I Co and 1 0.0025 mg/I Se 0.0010 mg/I V 0.0003 mg/I Na2EDTA.2H 20 2.5 mg/I Macro nutnients: Na2Si0 3. 91- 2 0 10.0 mg/I NaNO3 0.27 mg/I KH 2PO 4 0.14 mg/I K

2HP0 4 0.18 mg/I Vitamins: Thiamine 75.0 Pg/I B

12 1.0 Pg/I Biotin 0.75 pg/I
The hardness: 180 mg/I expressed as COC03 and the pH: 7.7 1 0.3. 6.4. Preparation oftest solutions The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film ofthe test substance on the water surface). The batch of CAI. Pigment Blue 61 tested was adark blue powderwith granules with apurity of 96.830% and not completely soluble intest medium at the loading rate prepared. Preparation of test solutions started with a loading rate of 100 mg/I applying a1 0-minute treatment period with ultrasonic waves followed by a 3days of magnetic stirring to reach maximum solubility of thetest substance inthe test medium. Subsequently, the aqueous mixture was filtered through amembrane filter (Schleicher &Schuell, 0.45 pm). The resulting filtrate was observed to be clear and light blue. 6.5. Limit test 6.5.1. Test concentrations 0.1. Pigment Blue 61 Filtered solution prepared at aloading rate of 100 mg/I Controls Test medium without test substance or other additives 6.5.2. Test procedure and conditions Test duration 48 hours Test type Static

-Page 9-

### c.I. Pigment Blue 61 NOTOX Project 488233 Test vessels 100 ml, all-glass Medium Adjusted ISO medium Number of daphnids 20 per concentration Loading 5per vessel containing 80 ml oftest solution Light 16 hours photoperiod daily Feeding No feeding Aeration No aeration ofthe test solutions introduction of daphnids Within 25 minutes after preparation of the test solutions 6.5.3. Sampling for analysis of test concentrations During the limit test samples for possible analysis were taken from both the control and the limit concentration according to the schedule below. Inaddition, the filter used for preparation of the limit concentration was stored for possible analysis of the residue. The method ofanalysis is described inthe appended Analytical Report (Appendix 11). Frequency att--0 hand t=48 h Volume 2 ml from the approximate centre of the test vessels Storage Samples were stored inafreezer until analysis. At the end ofthe exposure period, the replicates were pooled at each test group before sampling. Additionally, reserve samples of2 ml were taken for possible analysis. Ifnot used, these samples were stored inafreezer for a maximum of three months after delivery of the draft report, pending on the decision ofthe sponsor for additional analysis. 6.5.4. Measurements and recordings immobility (including mortality) At 24 hours and at 48 hours. pH and dissolved oxygen Atthe beginning and atthe end of the test, for both test groups. Temperature of medium Continuously inatemperature controlvessel, beginning atthe start of the test. 6.6. Electronic data capture Observations/measurements inthe study were recorded electronically using the following programme(s): REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA)

- Page 10-C.I. Pigment Blue 61 NOTOX Project 488233 6.7. Interpretation 6.7.1. Data handling The averageexposure concentration was calculated as 

## ]C_,.' _xC,, 4 8 being the geometric means of the concentrations ofC.l. Pigment Blue61 measured inthe samples taken atthe start 

### (Ct.0) and theend of the test (Ct 1 4). No ECsO could be calculated because the test substance proved to be non-toxic (EC 50> maximum soluble concentration tested). 6.7.2. Acceptability ofthe test 1. in the control, no daphnids became immobilised or trapped at the surface of the water. 2. The oxygen concentration at the end of the test was 2,3mg/l incontrol and test vessels. 6.8. List of deviations 6.8.1. List of protocol deviations There were no deviations from the protocol. 6.8.2. List of standard operating procedures deviations Any deviations from standard operating procedures were evaluated and filed inthe studyfile. There were no deviations from standard operating procedures that affected the integrity ofthe study.
7. RESULTS 7.1. Measured concentrations The results of analysis of the samples taken during the limit test are described inTable 2of the appended Analytical Report. Analysis of the samples taken at the start of the test showed ameasured concentration of 57.8 pg/I at the loading rate of 100 mg/I. During the exposure period the measured concentration decreased to 40.2 pg/I (i.e. 69% of initial). Based on these results, the average exposure concentration was calculated to be 48 pg/I. 7.2. Immobility Table 1shows the responses recorded during the limit test. No immobility of daphnids was observed ineither the limit concentration or the control group.

- Page 11

-

### C.l. Pigment Blue 61 NOTOX Project 488233
Table 1 Acute immobilisation ofdaphnids after 24 and 48 hours

Concentration Vessel Number Response at 24 h Response at48 h C.l. Pigment number Daphnia Total Total Blue 61 exposed number % number % control A 5 0 0 0 0 B5 0 0 C 5 0 0 D 5 0 0 48A 5 0 0 0 0 B5 0 0 C 5 0 0 D 5 0

__ _ 0 7.3. Determination of effect concentrations Table 2shows the effect parameters based on average exposure concentrations. Table 2 Effect parameters Parameter Concentration C.l. Pigment Blue 61 W11~) NOEC 48 24 & 48h-ECo >48 7.4. Experimental conditions The results of measurement of pH and oxygen concentrations (mg/I) are presented inTable 3. These test conditions remained within the limits prescribed by the protocol (pH:*6.0-8.5, not varying by more than 1.5 unit; oxygen: !3 mg/I at the end ofthe test). The temperature ofthe test medium was 20.7*C at the start ofthe test. The temperature continuously measured inatemperature control vessel varied between 20.9 and 21.3*C during the test, and complied with the requirements as laid down inthe protocol (1 8-22*C, constant within 2*C).
Table 3 pH and oxygen concentrations Concentration Start (t=G h) End t=8 h) C.l. Pigment Blue 61 (pig/) pH 02 pH 02 control 7.8 8.9 8.1 8.9

48 7.8 8.7 8.0 8.9
8. CONCLUSION Under the conditions ofthe present study C.l. Pigment Blue 61 did not induce acute immobilisation of Daphnia magna at 48 pg/I after 48 hours of exposure (NOEC). The 48h-EC5 owas >48 pg/I based on average exposure concentrations. This concentration was above the given water solubility limit of C.l. Pigment Blue 61 (i.e. 2.5 pg/I).

-Page 12

-C.I. Pigment Blue 61 NOTOX Project 488233
APPENDIX I REFERENCE TEST
Start: 24 June 2008 End :26 June 2008 48-hour Acute Toxicity Study inDaphnia magna with potassium dichromate (K2Cr 2 O

7 f) (NOTOX Project 488779). The study procedures described inthis report were based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation TesV', Adopted April 13, 2004, the EEC directive 92/69, Part C.2. "Acute toxicityfor Daphnia", December 1992, and the ISO International Standard 6341. The reference testwas carried out to check thesensitivity ofthe test system as used by NOTOX. Daphnia were exposed for amaximum of48 hours to K 2Cr 2 O

7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1. 8mg/I and to acontrol. Twenty daphnia were exposed per concentration. The reference substance, potassium dichromate (K 2Cr 2 O

7 ,,art. 4864, batch no. K34869764 607) was obtained from Merck, Darmstadt, Germany. Acute immobilization ofdaphnia after24 and48hours inthe reference test with potassium dichromate:

Concentration Number %immobile Expected response % (mg/I) Exposed 24h 48h After 48 hours Minimal Maximal control 20 0 0 0 102

0.10 20 0 0 0 10 0.18 20 0 0 0 10 0.32 20 0 5 0 30 0.56 20 5 15 0 100 1.0 20 80 100 40 100 1.8 20 100 100 100 100 B6ased -on historical data oftheprevious years (n>60). 2Amaximum response of 10% does not invalidatethe results ofthe test. The actual responses inthis reference testwith K 2Cr2O 7 are within the ranges ofthe expected responses at the different concentrations. Hence, the sensitivityof this batch ofD. magna was inagreement with the historical data collected at NOTOX. The 24h-EC 5 0 was 0.77 mg/I with a 95% confidence interval between 0.68 and 0.91 mg/. The 48h-EC5owas 0.82 mg/I with a 95% confidence interval between 0.53 and 0.77 mg/I. The raw data from thisstudy are kept inthe NOTOX archives. The test described above was performed under GLP with a QA-check.

-Page 13-

## APPENDIX 11

ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH C.I. PIGMENT BLUE 61 (STATIC); DETERMINATION OF THE CONCENTRATIONS

Author Ir. M.J.C. Brekelmans Study completion date

October21, 2008

Laboratory Proiect Identification NOTOX Project 488233 NOTOX Substance 183591/A

-Page 1of9-0.I. Pigment Blue 61 NOTOX Project 488233
1. CONTENTS 1. CONTENTS .............. .................... 2 2. REPORT APPROVAL.......................3 3. INTRODUCTION.........................4 3.1. Preface...........................4 3.2. Aim of the study............................................................................ 4 4. MATERIALS AND METHODS............................................. 4 4.1. Reagents.................................................................................... 4 4.2. Samples................................ ........................ 4 4.3. Analytical method ............................................................................ 5 4.3.1. Analytical conditions.................................................................. 5 4.3.2. Preparation ofthe calibration solutions.............................................. 5 4.3.3. Sample injections ..................................................................... 6 4.4. Electronic data capture....................................................................... 6 4.5. Formulas..........................6 5. RESULTS

............................. 7 5.1. Calibration curves.......................7 5.2. Samples..................................................................................... 7 5.2.1. Procedural recovery samples........................................................ 7 5.2.2. Test samples.......................................................................... 7 6. TABLES .................. ................................................................. 8 7. FIGURES........................................................................................... 9

-Page 2

-C.l. Pigment Blue 61 NOTOX Project 488233 2. REPORT APPROVAL
PRINCIPAL SCIENTIST: Ir.M.J.C. Brekelmans (Analytical Chemistry)



## Date: 

# C bc~eK U Z

-Page 3

-C.lI. Pigment Blue 61 NOTOX Project 488233 3. INTRODUCTION 3.1. Preface Study plan Start 23 September 2008 (analytical study) Completion 24 September 2008 3.2. Aim of the study The purpose of the analytical study was to determine the actual concentrations insamples taken from the test solutions used during the ecotoxicity test. 4. MATERIALS AND METHODS 4.1. Reagents Water Tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA) Methanol VWR International, Leuven, Belgium Sodium 1-heptanesulfonate Fluka Chemie, Buchs, Switzerland monohydrate (HSA) Aceticacid Merck, Darmstadt, Germany Dimethyl sulfoxide (DMSO) Merck ISO-medium see main report All reagents were of analytical grade, unless specified otherwise. 4.2. Samples The samples were stored inthe freezer. Storage stability ofsamples under these conditions was determined inNOTOX project 488235. On the day ofanalysis, the frozen samples were defrosted at room temperature. The test samples were diluted ina 1:1 (v:v) ratiowith methanol and analysed. Concentrations inthe test solution were expected to be low. To demonstrate proper preparation of the test solution, itwas decided to analyse the residue on afilter used during preparation of this solution. An aliquot of the residue was transferred quantitatively into a 5ml volumetric:flask using DMSO. This solution was ultrasonicated for 10 minutes, diluted by afactorof 100 with 50/50 (vlv) methanol/ISO-medium and analysed.

-Page 4

-

### C.I. Pigment Blue 61 NOTOX Project488233

4.3. Analytical method 4.3.1. Analytical conditions Quantitative analysis was based on the analytical method validated forthe test substance in NOTOX project 488235. Analyticalconditions: Instrument Alliance Separation Module 2695 (Waters, Milford, MA, USA) Detector Dual A Absorbance Detector 2487 (Waters) Column Symmetry Shield C18, 150 mm x4.6 mm iW., dp =5 pm (Waters) Column temperature 5o*C 

### ± i*C Injection volume 100 PI Mobile phase A

- 5/0.5/94.5 (vlv/v) water/acetic acid/methanol, containing 1.2 g/l HSA B

- 5/0.5/94.5 (v/v/v) water/acetic acid/[50/50 (v/v) methanol/water], containing 1.2 g/l HSA C

- methanol Gradient Time %A %B %C [minutes]

______ _____ 0 0 100 0 20 100 0 0 

### 20.1 0 100 0 23 0 100 0 23.1 0 0 100

28 0 0 100 28.1 0 100 0

35 0 100 0 Flow 2mI/mmn UVdetection 313 nm 4.3.2. Preparation ofthe calibration solutions Stock- and spikingj solutions Stock solutions ofthe test substance were prepared indimethyl sulfoxide at concentrations of 985 and 1240 mg/I. Inorder to dissolve the test substance the solutions were ultrasonicated for 10 minutes. Spiking solutions were made upfrom astock solution and/or dilutions of this solution. The solvent of the spiking solutions was dimethyl sulfoxide. Calibration solutions Calibration solutions inthe concentration range 0.04
- 10 mg/I were prepared from two stock solutions. The end solution of the calibration solutions was 50/50 (vlv) methanol/water. Procedural recovery samples 2 ml blank medium was spiked with the test substance at atarget concentration of 0.1 or 10 mg/I. The accuracy samples were treated similarly as the test samples (see paragraph 4.2 'Samples').

-Page 5

-

### C.I. Pigment Blue 61 NOTOX Project 488233 4.3.3. Sample injections Calibration solutions were injected induplicate. Test samples and procedural recovery samples were analysed by single injection. 4.4. Electronic data capture System control, data acquisition and data processing were performed using the following programme:

- Empower version 5.00 (Waters, Milford, MA, USA). Temperature and/or relative humidity during sample storage and/or performance ofthe studies were monitored continuously using the following programme:

- REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific,Trenton,

NJ, USA). 4.5. Formulas Response (R) Peak area test substance [units] Calibration curve R=aCN + b where: CN = nominal concentration [mg/I] a = slope [units xI/mg] b = intercept [units] 

### Analysed concentration (CA) 

### CA = (R-b)xd [mg/I] a where: d = dilution factor Recovery CA .10% CN
Relative to initial concentration CA (t=xhours) x100% CA (t=O0hours)

-Page6-

### C.I. Pigment Blue 61 NOTOX Project 488233 5. RESULTS 5.1. Calibration curves Several peaks were observed inthe chromatogram ofthe testsubstance solution. The peak area of the largest peak (i.e. the peak at aretention time of 10 minutes) was used as response inthe calculations. A calibration curve was constructed using four concentrations. For each concentration, two responses were used.2Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. The coefficient of correlation (r)was> 0.99. Representative chromatograms of atest substance solution and blank solution are shown in Figure 1. 5.2. Samples 5.2.1. Procedural recovery samples The results forthe procedural recovery samples are given inTable 1. Mean recoveries ofthe procedural recovery samples were 101% and 102%. Because the criterion that mean recoveries should be between 70% and 110% was met, the results forthe test samples were accepted. 5.2.2. Test samples The results for the test samples are given inTable 2. Inorder to demonstrate proper preparation ofthe test solution, itwas decided to analyse the residue on the 0.45 pm filter used for preparation ofthe nominal 100 mg/I test solution. Based on retention times of the peaks inthe chromatogram, the residue was identified as test substance indicating proper preparation ofthe test solution.

-Page 7

-C.l. Pigment Blue 61 NOTOX Project488233 6. TABLES Table I Procedural recovery samples D-1ate of Date of Target Nominal Analysed Recovery Mean preparation analysis concentration concentration concentration recovery Idd-mm-yyl [dd-mm-yy] [mg/I] [mg/l [mg/Il 1%) [%]

23-09-08 23-09-08 0.1 0.100 0.0990 99 102 0.100 0.104 104 23-09-08 23-09-08 10 10.0 10.1 101 101 10.0 10.1 101

Table 2 Concentrations of the test substance in test medium (limit test) Time of Date of Date of Concentration sampling sampling analysis Loading rate Analysed Relative to

[hours] [dd-mm-yy] [dd-mm-yy] [mg/I] [mg/I] initial (%l 0 15-09-08 23-09-08 0 n.d. 100 0.05781 48 17-09-08 23-09-08 0 nd. 100 0.04021 69 Samples were stored inthe freezer until the day of analysis. Afiltered test solution prepared at the loading rate Obtained by extrapolation of the calibration curve. Result isindicative. n.d. Notdetected.

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-C.I. Pigment Blue 61 NOTOX Project 488233 7. FIGURES

Figure I Chromatograms of a 10.0 nig/ test substance solution [top; res. id. 1526] and corresponding blank [bottom; res. id. 1502].

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