## FINAL REPORT

Study Title



## DEVELOPMENT AND VALI DATION OF

AN ANALYTICAL METHOD FOR THE ANALYSIS OF

CASTOR OIL, SULFATED , SODIUM SALT, 75% I N WATER

Author

I. van Doormalen

- Schaap, BSc

.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 503334

Substance 205003/A

- Page 1 of 11

-Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

Final re port

- Page 2 - 1. CONTENTS

1. CONTENTS

................................................................................................ ...................................... 2

2. STATEMENT OF GLP COM PLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE ST ATEMENT

............................................................................................ 4

4. SUMMARY

................................................................................................ ........................................ 5

5. INTRODUCTION

.............................................................................................................................. 6

5.1. Preface

................................................................................................ ................................... 6

5.2. Aim of the study

...................................................................................................................... 6

5.3. Storage and retention of records and materials

..................................................................... 6

6. MATERIALS

................................................................................................ ...................................... 7

6.1. Test substance

....................................................................................................................... 7

6.2. Electronic data capture

........................................................................................................... 7

6.3. List of deviations

..................................................................................................................... 7

7. VALIDATION OF AN ANA LYTICAL METHOD

................................................................................. 8

7.1. Guidelines

............................................................................................................................... 8

7.2. Statement

............................................................................................................................... 8

8. CARBON CONTENT

........................................................................................................................ 9

8.1. Reagents

................................................................................................ ................................ 9

8.2. Performance of the study

....................................................................................................... 9

8.3. Analytical method

................................................................................................................... 9

8.4. Results

................................................................................................ ................................ . 10

8.5. Conclusion

........................................................................................................................... 10

APPENDICES

Appendix 1 Certificate of analysis

........................................................................................................ 11Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

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- Page 5

- 4. SUMMARY

Castor Oil, Sulfated, Sodium Salt, 75% is a complex mixture containing many sulfated and non

sulfated components.

A general method is considered the best option for the analytical support of ecotoxicity studies with

daphnids and algae and for the analytical support of a combined 28

- day repeated dose toxicity study.

Upon request of the sponsor, feasibility of SO 3 content determination by titration was tested. The

method which was successfully used for determina tion of the SO 3 content in the test substance itself,

proved not sufficiently sensitive to support ecotoxicity studies in the concentration range of 1

- 100 mg/l.

Total Organic Carbon (TOC) wa s considered to be a good alternative , s ince the test substance

d issolves in aqueous solutions up to 200 mg/ml. TOC was therefore used for analysis of the samples

fr om ecotoxicity studies with daphnids and algae and from the combined 28

- day repeated dose toxicity

study

,

TOC analysis is a technique that does not require test substance specific validation. Therefore

validation of an analytical method was omitted. Instead TOC content of the test substance was

determined in order to convert concentrations from mg C/l measured to mg test substance/l.Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

Final re port

- Page 6

- 5. INTRODUCTION

5.1. Preface

Sponsor Sulfated Castor Oil Consortium, Society of Chemical

Manufacturers & Affiliates

1850 M Street NW, Suite 700

Washington, DC 20036

USA

Study Monitor Rodney Boatman

PCA Services

2704 Trail Wood Drive

Durham, NC 27705

USA

Test Facility WIL Res earch Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director I. van Doormalen

- Schaap, BSc

.

Study Plan Start : 15 October 201 3

Completion : 0 3 December 201 3

5.2. Aim of the study

The aim of the study was to develop a nd to validate an analytical method for the quantitative analysis

of Castor Oil, Sulfated, Sodium Salt, 75% in water

, ISO

- medium, M2

- medium and M7

- medium vehicle

, to support the ecotoxicity studies with daphnids and algae and the combined 28

- day repeated d ose

toxicity study.

5.3. 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 WIL Re search

Europe 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. WIL

Research Europe will retain information concerning decisions made.

Those specimens requiring refrigeration or freezing will be retained by WIL Research Europe for as

long as the quality of the specimens permits evaluation but no longer than three months after

finalization of the report.

WIL Research Euro pe 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.Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

Final re port

- Page 7

- 6. MATERIALS

6.1. Test substance

6.1.1. Test substance information

Identification Castor Oil, S ulfated, Sodium Salt, 75%

CAS Number 68187

- 76

- 8

Description Clear amber liquid

Batch R

- 10135

- 064

Purity/Composition 7 4

. 4 %

Test substance storage At room temperature in the dark

Stable under storage conditions until 18 July 2014 (expiry date)

The c ertificate of analysis is shown in Appendix 1

.

6.1.2. Study specific test substance information

Purity/composition correction factor required Yes, correction factor is 1. 34 (7 4

. 4 % purity)

6.2. Electronic data capture

System control, data acquisition and data proces sing were performed using the following programme:

Shimadzu TOC

- Controle V version 2. 10 (Shimadzu , Kyoto, Japan)

Temperature, relative humidity and/or atmospheric pressure during sample storage and/or

performance of the studies was monitored continuously using the following programme:

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

USA)

6.3. List of deviations

6.3.1. List of protocol deviations

There were no deviations from the protocol.

6.3.2. List of standard operating proced ures deviations

There were no deviations from standard operating procedures (SOPs).Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

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- Page 8

- 7. VALIDATION OF AN ANA LYTICAL METHOD

7.1. Guidelines

European Commission: Guidance for Generating and Reporting Methods of Analysis in Support of

Pre

- registration Data Requireme nts for Annex II (Part A, section 4) and Annex III (Part A, section 5) of

Directive 91/414, SANCO/3029/99 rev. 4 (11/07/00).

7.2. Statement

Castor Oil, Sulfated, Sodium Salt, 75% is a complex mixture containing many sulfated and non

sulfated components.

Ba sed on the complexity of the test substance and based on experience, d evelopment of a

chromatographic method for analysis of all individual compounds is presumed not possible.

Gas chromatography fails since the test substance contains compound s with a rela tively high

molecular weight and of which part of it are salts . Liquid chromatography (HPLC) with

spectrophotometric detection fails since the compounds in the test substance do not contain

chromophoric groups. HPLC with refractive index detection is not s ufficiently sensitive to measure

concentrations in the concentration range of 1

- 100 mg/l i.e. the concentration range to be used in the

ecotoxicity studies. HPLC with mass spectrometric detection fails on the differences in ionization of the

various compou nds in the test substance and on ion

- suppression of co

- eluting compounds. Full

separation of this complex mixture is not possible.

Since also s pectroscopy is not an alternative since the compounds in the test substance do not

contain chromophoric groups

, a general method is considered the best alternative for the analytical

support of ecotoxicity studies with daphnids and algae and for the analytical support of a combined 28

- day repeated dose toxicity study. Upon request of the sponsor, feasibility of SO 3 content determination

by titration, which is a general method, was tested. The method which was successfully used for

determination of the SO 3 content in the test substance itself, proved not sufficiently sensitive in the

concentration range of 1

- 100 mg/l ( results a rchi ved in the raw data)

.

Since the test substance dissolves in aqueous solutions up to 200 mg/ml

, analysis of Total Organic

Carbon (TOC) which is another general method wa s considered to be a good alternative for the

analysis of samples from t he above mentioned studies

.

TOC analysis is a technique that do es not require test substance specific validation. Therefore

validation of an analytical method was omitted. Instead TOC content of the test substance was

determined in order to convert conce ntrations from mg C/l me asured to mg test substance

.Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

Final re port

- Page 9

- 8. CARBON CONTENT

8.1. Reagents

Sodium carbonate Merck, Darmstadt, Germany

Art.nr. 1.06649, Batch A0110492944.

Glucose Sigma

- Aldrich, Steinheim, Germany.

Art. Nr. 67528, Batch 108k00301

All reagents were of analytical grade purity, unless specified otherwise.

8.2. Performance of the study

For quantitative analysis of the test substance in aqueous solutions, Total Organic Carbon (TOC)

analysis in the Non

- Purgable Organic Carbon (NPOC) mode was used. In t his mode, volatile

compounds were not included.

This analytical technique does not require test substance specific validation. To calculate test

substance concentration from the measured carbon concentration, the carbon content of the test

substance was d etermined.

Four samples (i.e. two samples for total carbon content and two samples for inorganic carbon content)

of the test substance were taken for determination of the Total Organic Carbon (TOC) content.

Calibration standards were Glucose as total carb on (TC) standard and Sodium carbonate as inorganic

carbon (IC) standard.

8.3. Analytical method

8.3.1. Analytical conditions

Instrument TOC

- V CPH total organic carbon analyzer with SSM

- 5000A

(Solid Sample module for Total Organic Carbon Analyzer)

(Shimadzu, Kyoto, J apan)

Number of repeats 1

8.3.2. Sample measurements

The performance of the TOC analyzer was checked using Glucose and Sodium carbonate, duplicate

samples were accurately weighed and analyzed once

. The analytical system was considered suited

for use when the car bon content of g lucose (i.e. 40% carbon) was within 38

- 42% and the carbon

content sodium carbonate (i.e. 56.6% carbon) was within 53.8

- 59.4%.

Duplicate test substance samples were accurately weighed and w ere analyzed once

.Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

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- Page 10

- 8.4. Results

The carbon content results of glucose were 39.92 and 41.77%. The carbon content results of sodium

carbonate were 5 3.85 and 55.38%. Since the results were within the criteria, the analytical system was

considered suited for determination of the Total Organic Carbon of the test substance

.

The results of the total carbon (TC) en inorganic carbon (IC) content of the test substance are given in

Table 1

. The Total Organic Carbon (TOC) is defined as the total carbon content minus the i norganic

carbon content. The Total Organic Carbon is 54.48 %.

Table 1 The total and inorganic carbon content of test substance

TC content IC content

Sample 1 53.17 % 0. 04 %

Sample 2 55.83 % 0.00%

Mean 5 4 .50 % 0.02 %

8.5. Conclusion

The Total Organic Carbon (TOC) content of the test substance was determined to be 54.48 %.Castor Oil, Sulfated, Sodium Salt, 75% Project 503334

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- Page 11

- Appendix 1 Certificate of analysis