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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 

PREVENTION, PESTICIDES AND 

TOXIC SUBSTANCES 

Subject: Follow-up to fax transmission of 4/13/12 (ePMA removal ofCBI claims regarding 

identified studies) 

Dear Mr. Merritt: 

This is sent to you pursuant our conversation of several minutes ago. In Larry Robinson's fax of 

April 13th while there is a waiver of confidential business information (CBI) claims in the 

identified studies there is a an assertion of copyright protections and some additional text that 

provides in part ... "Reproduction, reference, summarization, retransmission or other use of this 

copyrighted document. .. including electronic transmission, using the internet or email .... is 

strictly prohibited .... " 

While we understand the basis for the copyright protections, we have a concern that the language 

in the fax might be interpreted to mean that ePMA is asserting that the studies submitted per the 

TSCA activity are not to be made available in an Agency e-docket (electronic docket) or be 

included in EPA websites among the collection of TSCA health 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 of non-CBI materials submitted by entities subject to TSCA 

regulations (including the posting of such materials on government websites) and submitted by 

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 of the studies. 

Can you please confirm CPMA's intent? Many thanks. 

Scott M. Sherlock, Attorney Advisor 

Environmental Assistance Division 

Office of Pollution 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 1 of 22-
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 of the 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 specific test 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 of test solutions ................................................................. 9

6.5. Limit test ..................................................................................... 9

6.5.1. Test concentrations ................................................................... 9

6.5.2. Test procedure and-conditions....................................................... 9

6.5.3. Sampling for analysis of test concentrations ...................................... 10

6.5.4. Measurements and recordings ..................................................... 10

6.6. Electronic data capture..................................................................... 10

6.7. I nterpretation ................................................................................ 11

6.7.1. Data handling ........................................................................ 11

6.7.2. Acceptability of the test.............................................................. 11

6.8. List of deviations ............................................................................ 11

6.8.1. List of protocol deviations........................................................... 11

6.8.2. List of 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 Project 488233

2. STATEMENT OF GLP COMPLIANCE

NOTOX B..'s-Hertogenbosch, The Netherlands

The study described in this report has been correctly reported and was conducted in compliance

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 is responsible for Good Laboratory Practice (GLP) compliance for all test

substance information unless determined by NOTOX.

NOTOX B.V.

Ir. L.M. Bouwman Ing. E.J. van de Waart, M.Sc.

Study Director Head of In Vitro & 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 Quality Assurance Unit to confirm that the methods

and results accurately and 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 is the date of reporting to the Study Director. The QAU report was then

forwarded to the 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 0 1-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 Project 488233

4. SUMMARY

Acute Toxicity Study in Daphnia magna with C.l. Pigment Blue 61.

The study procedures described in this report were based on the OECD guideline No. 202,

2004. In addition, 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 a dark blue powder with granules with a purity of

96.830% and not completely soluble in test medium at the loading rate prepared.

Preparation of test solutions started with a loading rate of 100 mg/I applying a 10-minute

treatment period with ultrasonic waves followed by a 3 days of magnetic stirring to reach

maximum solubility of the test substance in the test medium. Subsequently, the aqueous

mixture was filtered through a membrane filter (0.45 pm). The resulting filtrate was observed to

be clear and light blue.

A limit test was performed, exposing twenty daphnids per concentration (four replicates, five

daphnids each) to the filtered solution prepared at a loading rate of 100 mg/I and a control

group. The total test period was 48 hours and samples for analytical confirmation of actual

exposure concentrations were taken at the start and the end of the test.

Analysis of the samples taken at the start of the test showed a measured 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 by the protocol and was considered valid.

0.1. Pigment Blue 61 did not induce acute immobilisation of Daphnia magna at 48 pg/I after 48

hours of exposure (NOEC).

The 48h-EC50 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 Project 488233

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 of the study

The purpose of the study was to evaluate the test substance for 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 EC5o at 24 and 48 hours of exposure.

5.3. Guidelines

The study procedures described in this report were based on the Organization for Economic Cooperation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No.

202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004.

In addition, 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 of the

mobility of Daphnia magna Straus - Acute toxicity test, Third edition, 1998-04-01.

* Guidance document on aquatic toxicity testing 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 in the NOTOX

archives for a period of at least 2 years after finalization of the report. After this period, the

sponsor will be contacted to determine 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 quality of the specimens permits evaluation but no longer than three months after finalization

of the report.

NOTOX will retain a test substance sample until the expiry date, but no longer than 10 years

after finalization of the report. After this 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 is the concentration of test substance estimated to immobilise 50% of the daphnids

after a defined period of exposure.

No Observed Effect Concentration (NOEC) is the highest concentration tested at which no

effect (i.e. immobilisation) is recorded.

6. MATERIALS AND METHODS

6.1. Test Substance

6.1.1. Test substance information

Identification C.l. Pigment Blue 61

Structure

N

OH~

molecular formula C,,H29N,03S

Molecular weight 596

CAS Number 1324-76-1

Description Dark blue powder with granules

Batch 2084701

Purity 96.830%

Test substance storage At room temperature in the dark

stability under storage conditions Stable

Expiry date 02 January 2018

6.1.2. Study specific test substance information

Stability in water At least 96 hours

Solubility in water 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 in water (bi dest.) at ambient temperature is 2.5 pg/I (Clariant, SU-Technology & Innovation Management, Pigment Analytics).

6.2. Test system

Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical

parthenogenesis under specified breeding conditions.

Source In-house laboratory culture with a known history.

Reason for selection This system has been selected as an internationally

accepted invertebrate species.

Validity of batch Daphnids originated from a healthy stock, 2nd to 5th

brood, showing no signs of stress such as mortality

>20%, presence of males, ephippia or discoloured

animals and there was no delay in the production of

the first brood.

Characteristics For the test selection of young daphnids with 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 3 days old, by

placing about 250 of them into 5 litres of medium in an

all-glass culture vessel.

Maximum age of the cultures 4 weeks

Renewal of the cultures After 7 days of cultivation half of the medium twice a

week.

Temperature of medium 18-22'C

Feeding Daily, a suspension of fresh water algae.

Medium M7, as prescribed by Dr. Elendt-Schneider

(Elendt, B.-P., 1990: Selenium deficiency in

Crustacea. An ultrastructural approach to antennal

damage in Daphnia magna Straus. Protoplasma 154,

25-33).

Composition of medium M7:

Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse

osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

Macro salts: 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 to freshly 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. 91-20 10.0 mg/I

NaNO3 0.27 mg/I

KH 2PO4 0.14 mg/I

K2HP0 4 0.18 mg/I

Vitamins: Thiamine 75.0 Pg/I

B12 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 of test 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 of the test substance on the water surface).

The batch of CAI. Pigment Blue 61 tested was a dark blue powder with granules with a purity of

96.830% and not completely soluble in test medium at the loading rate prepared.

Preparation of test solutions started with a loading rate of 100 mg/I applying a 1 0-minute

treatment period with ultrasonic waves followed by a 3 days of magnetic stirring to reach

maximum solubility of the test substance in the test medium. Subsequently, the aqueous

mixture was filtered through a membrane 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 a loading 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 5 per vessel containing 80 ml of test solution

Light 16 hours photoperiod daily

Feeding No feeding

Aeration No aeration of the 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. In addition, the filter used for preparation of the

limit concentration was stored for possible analysis of the residue. The method of analysis is

described in the appended Analytical Report (Appendix 11).

Frequency at t--0 h and t=48 h

Volume 2 ml from the approximate centre of the test vessels

Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each test group before

sampling.

Additionally, reserve samples of 2 ml were taken for possible analysis. If not used, these

samples were stored in a freezer for a maximum of three months after delivery of the draft

report, pending on the decision of the sponsor for additional analysis.

6.5.4. Measurements and recordings

immobility (including mortality) At 24 hours and at 48 hours.

pH and dissolved oxygen At the beginning and at the end of the test, for both

test groups.

Temperature of medium Continuously in a temperature control vessel,

beginning at the start of the test.

6.6. Electronic data capture

Observations/measurements in the 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 average exposure concentration was calculated as ]C_,.' _xC,, 48 being the geometric

means of the concentrations of C.l. Pigment Blue 61 measured in the samples taken at the start

(Ct.0) and the end of the test (Ct14).

No ECsO could be calculated because the test substance proved to be non-toxic (EC50 >

maximum soluble concentration tested).

6.7.2. Acceptability of the 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, 3 mg/l in control 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 in the study file.

There were no deviations from standard operating procedures that affected the integrity of the

study.

7. RESULTS

7.1. Measured concentrations

The results of analysis of the samples taken during the limit test are described in Table 2 of the

appended Analytical Report.

Analysis of the samples taken at the start of the test showed a measured 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 1 shows the responses recorded during the limit test. No immobility of daphnids was

observed in either the limit concentration or the control group.

- Page 11 -
C.l. Pigment Blue 61 NOTOX Project 488233

Table 1 Acute immobilisation of daphnids after 24 and 48 hours

Concentration Vessel Number Response at 24 h Response at 48 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 2 shows 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 in Table 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 of the test).

The temperature of the test medium was 20.7*C at the start of the test. The temperature

continuously measured in a temperature control vessel varied between 20.9 and 21.3*C during

the test, and complied with the requirements as laid down in the 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 of the 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-EC 5o was > 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 in Daphnia magna with potassium dichromate (K2Cr2O7f) (NOTOX

Project 488779).

The study procedures described in this 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 toxicity for Daphnia", December 1992, and the ISO International Standard 6341.

The reference test was carried out to check the sensitivity of the test system as used by

NOTOX. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0. 10,

0. 18, 0.32, 0.56, 1.0 and 1. 8 mg/I and to a control. Twenty daphnia were exposed per

concentration.

The reference substance, potassium dichromate (K2Cr2O7,, art. 4864, batch no. K34869764 607)

was obtained from Merck, Darmstadt, Germany.

Acute immobilization of daphnia after 24 and 48 hours in the 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 of the previous years (n>60).

2 A maximum response of 10% does not invalidate the results of the test.

The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected

responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was

in agreement with the historical data collected at NOTOX.

The 24h-EC 50 was 0.77 mg/I with a 95% confidence interval between 0.68 and 0.91 mg/.

The 48h-EC5o was 0.82 mg/I with a 95% confidence interval between 0.53 and 0.77 mg/I.

The raw data from this study are kept in the 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

October 21, 2008

Laboratory Proiect Identification

NOTOX Project 488233

NOTOX Substance 183591/A

-Page 1of 9-
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 of the 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 in samples 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)

Acetic acid 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 in the freezer. Storage stability of samples under these conditions was

determined in NOTOX project 488235. On the day of analysis, the frozen samples were defrosted

at room temperature. The test samples were diluted in a 1:1 (v:v) ratio with methanol and

analysed.

Concentrations in the test solution were expected to be low. To demonstrate proper preparation

of the test solution, it was decided to analyse the residue on a filter used during preparation of

this solution. An aliquot of the residue was transferred quantitatively into a 5 ml volumetric: flask

using DMSO. This solution was ultrasonicated for 10 minutes, diluted by a factor of 100 with

50/50 (vlv) methanol/ISO-medium and analysed.

-Page 4 -
C.I. Pigment Blue 61 NOTOX Project 488233

4.3. Analytical method

4.3.1. Analytical conditions

Quantitative analysis was based on the analytical method validated for the test substance in

NOTOX project 488235.

Analytical conditions:

Instrument Alliance Separation Module 2695

(Waters, Milford, MA, USA)

Detector Dual A Absorbance Detector 2487 (Waters)

Column Symmetry Shield C18, 150 mm x 4.6 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 2 mI/mmn

UV detection 313 nm

4.3.2. Preparation of the calibration solutions

Stock- and spikingj solutions

Stock solutions of the test substance were prepared in dimethyl sulfoxide at concentrations of

985 and 1240 mg/I. In order to dissolve the test substance the solutions were ultrasonicated for

10 minutes.

Spiking solutions were made up from a stock solution and/or dilutions of this solution. The solvent

of the spiking solutions was dimethyl sulfoxide.

Calibration solutions

Calibration solutions in the 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 a target concentration of 0.1 or 10 mg/I.

The accuracy samples were treated similarly as the test samples (see paragraph 4.2 'Samples').

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C.I. Pigment Blue 61 NOTOX Project 488233

4.3.3. Sample injections

Calibration solutions were injected in duplicate. 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 of the 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 = a CN + b

where:

CN = nominal concentration [mg/I]

a = slope [units x I/mg]

b = intercept [units]

Analysed concentration (CA) CA = (R -b) x d [mg/I] a

where:

d = dilution factor

Recovery CA .10% CN

Relative to initial concentration CA (t = x hours) x 100% CA (t =O0hours)

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C.I. Pigment Blue 61 NOTOX Project 488233

5. RESULTS

5.1. Calibration curves

Several peaks were observed in the chromatogram of the test substance solution. The peak area

of the largest peak (i.e. the peak at a retention time of 10 minutes) was used as response in the

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 a test substance solution and blank solution are shown in

Figure 1.

5.2. Samples

5.2.1. Procedural recovery samples

The results for the procedural recovery samples are given in Table 1.

Mean recoveries of the procedural recovery samples were 101% and 102%. Because the

criterion that mean recoveries should be between 70% and 110% was met, the results for the test

samples were accepted.

5.2.2. Test samples

The results for the test samples are given in Table 2.

In order to demonstrate proper preparation of the test solution, it was decided to analyse the

residue on the 0.45 pm filter used for preparation of the nominal 100 mg/I test solution. Based on

retention times of the peaks in the chromatogram, the residue was identified as test substance

indicating proper preparation of the test solution.

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C.l. Pigment Blue 61 NOTOX Project 488233

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 in the freezer until the day of analysis.

A filtered test solution prepared at the loading rate

Obtained by extrapolation of the calibration curve. Result is indicative.

n.d. Not detected.

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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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