## FINAL REPORT

Study Title



## DETERMINATION OF PHY SICO

- CHEMICAL PROPERTIES OF

CASTOR OIL, SULFATED , SODIUM SALT, 75%

 Melting and boiling temperature

 Water content Karl Fis c her

 Content Sodium Sulphate

 Content SO 3

 Purity / composition

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 503335

Substance 205003 / A

- Page 1 of 27

-Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE ST ATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

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

5.1. Preface

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

5.2. Aim of the study

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

5.3. Storage and retention of records and materials

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

6. MATERIALS

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

6.1. Test substance

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

6.2. Electronic data capture

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

6.3. List of deviations

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

7. MELTING AND BOILING TEMPERATURE

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

7.1. Guidelines

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

7.2. Performan ce of the test

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

7.3. Analytical method

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

7.4. Interpretation

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

7.5. Res ults

................................................................................................ ................................ . 11

7.6. Conclusion

........................................................................................................................... 12

7.7. Figures

................................................................................................ ................................ . 12

8. WATER CONTENT KARL FISCHER

............................................................................................ 15

8.1. Reagents

............................................................................................................................. 15

8.2. Performance of the test

....................................................................................................... 15

8.3. Analytical method

................................................................................................................ 16

8.4. Interpretation

........................................................................................................................ 16

8.5. Results

................................................................................................ ................................ . 17

8.6. Conclusion

........................................................................................................................... 17

9. CONTENT SODIUM SULPH ATE

.................................................................................................. 18

9.1. Guidelines

............................................................................................................................ 18

9.2. Reagents

............................................................................................................................. 18

9.3. Reagent solutions

................................................................................................................ 18

9.4. Performance of the study

.................................................................................................... 18

9.5. Analytical method

................................................................................................................ 19

9.6. Interpretation

........................................................................................................................ 20

9.7. Results

................................................................................................ ................................ . 21

9.8. Conclusion

........................................................................................................................... 22

10. CONTENT SO 3

.............................................................................................................................. 23

10.1. Guidelines

............................................................................................................................ 23

10.2. Reagen ts

............................................................................................................................. 23

10.3. Reagent solutions

................................................................................................................ 23

10.4. Performance of the study

.................................................................................................... 23

10.5. Analytical method

................................................................................................................ 24

10.6. Interpretation

........................................................................................................................ 24

10.7. Results

................................................................................................ ................................ . 25

10.8. Conclusion

........................................................................................................................... 26

11. PURITY / COMPOSITION

............................................................................................................. 26Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 3 - TABLES

Table 1 Calibration of Karl Fischer apparatus

................................................................................ 17

Table 2 Water content of the solvent solution

................................................................................ 17

Table 3 Water content of the test substance

................................................................................. 17

Table 4 Content Sodium Sulphate

................................................................................................ . 21

Table 5 Relative standard deviation

............................................................................................... 21

Table 6 Accuracy samples

............................................................................................................. 22

Tabl e 7 SO 3 content

....................................................................................................................... 25

Table 8 Relative standard deviation

............................................................................................... 25

Table 9 Accuracy samples

............................................................................................................. 26

FIGURES

Figure 1 TGA curve of the test substance.

...................................................................................... 12

Figure 2 DSC curve of the test substance obtained with Experiment 1.

......................................... 13

Figure 3 DSC curve of the test substance obtained with Experiment 2.

......................................... 13

Figure 4 DSC curve of the test substance obtained with Experiment 3.

......................................... 14

APPENDICES

Appendix 1 Certificate of analysis

........................................................................................................ 27Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 6

- 4. SUMMARY

The results of the physico

- chemical properties of the test substance are given below.

Parameter Guideline(s) Result Comment

Melting temperature EC A.1

OECD 102

OPPTS 830.7200 - 4 °C ( 269 K) most likely due to

water in the test

substance

Boiling temperature EC A.2

OECD 103

OPPTS 830.7220 113 °C ( 386 K) most likely due to

water in the test

substance

Water content

- 24.3% w/w by Karl Fis c her

coulometric titration

Content Sodium Sulphate FAO

SANCO/3030/99 1.2 5 % w/w colorimetric titration

Content SO 3 FAO

SANCO/3030/99 5.58% w/w colorimetric titration

Purity / composition

- 74. 4 % (100 % minus water

and sodium sulphate

content )Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 7

- 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 Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director I. van Doormalen

- Schaap, BSc

.

Study Plan Start : 12 October 201 3

Completion : 07 November 201 3

5.2. Aim of the study

The aim of the study was to determine the following physico

- chemical properties for Castor Oil,

Sulfated, Sodium Salt, 75%.

 Melting and boiling temperature

 Water content Karl Fis c her

 Content Sodium Sulphate

 Content SO 3

 Purity / composition

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 Research

Europe archives for a per iod 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 Europe 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 503335

Final report

- Page 8

- 6. MATERIALS

6.1. Test substance

6.1.1. Test substance information

Identification Castor Oil, Sulfated, Sodium Salt, 75%

CAS Number 68187

- 76

- 8

Description Clear amber liquid

Batch R

- 10135

- 064

Purity/Composition To be determined in this project

Test substance storage At room temperature in the dark

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

The certificate of analysis is shown in Appendix 1.

6.1.2. Study specific test substance information

Purity/composition correction factor required No

6.2. Electronic data capture

System control, data acquisition and data processing were performed using the following pr ogramme:

- Advantage Integrity Software version 3.0 (TA Instruments, New Castle, DE, USA)

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

performance of the studies w as monitored continuously using the following programme s :

- 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 procedures deviations

Any deviations from standard operating procedures (SOPs) were evaluated and filed in the study file.

There were no deviations from SOPs that affected the integrity of the study.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 9

- 7. MELTING AND BOILING TEMPERATURE

7.1. Gu idelines

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of Physico

Chemical Properties, Guideline A.1: “Melting/Freezing Temperature”, Official Journal of the European

Union no. L142, May 31, 2008.

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of Physico

Chemical Properties, Guideline A.2: “Boiling Temperature”, Official Journal of the European Union no.

L142, May 31, 2008.

Organization for Economic Co

- operation and Development (OECD), OEC D Guidelines for the Testing

of Chemicals no. 102: "Melting Point / Melting Range", July 27, 1995.

Organization for Economic Co

- operation and Development (OECD), OECD Guidelines for the Testing

of Chemicals no. 103: "Boiling Point", July 27, 1995.

United States Environmental Protection Agency (EPA), Product Properties Test Guidelines no.

OPPTS 830.7200: "Melting Point/Melting Range", March 1998.

United States Environmental Protection Agency (EPA), Product Properties Test Guidelines no.

OPPTS 830.72 20: "Boiling Point/Boiling Range", August 1996.

7.2. Performance of the test

The melting and boiling temperature of the test substance were determined using differential scanning

calorimetry (DSC). This technique records the difference in heat flow of two cruc ibles, one filled with

test substance and the other left empty as reference , while these crucibles are subjected to a

controlled temperature programme. When the test substance undergoes a transition, such as melting

or evaporation, is indicated by a deviat ion from the base line of the heat

- flow record. Because melting

and evaporation are processes in which heat is consumed, for these processes the deviation from the

base line is in the endothermic direction. The melting temperature or boiling temperature (i f any) of the

test substance is evaluated from the DSC curve as the extrapolated onset temperature of the

endothermic melting or evaporation peak.

The test substance is a liquid at room temperature. To determine the melting temperature

, the test

substanc e was cooled to

- 9 0°C and then heated

.

As a safety precaution for the DSC cell a preliminary test was performed using a thermogravimetric

analyzer (TGA).

7.2.1. Preliminary test

11.0 mg test substance was heated at a rate of 20°C/minute from 25°C to 431°C. At this point 70 %

weight loss of the test substance was observed. After the experiment, the sample was cooled to and

equilibrated at 50 °C and the consistency of the test substan ce was determined.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 10

- 7.2.2. Main study

An overview of the experimental conditions of the main study is given below.

Experiment 1 2 3 4

Sample amount (mg) 11.16 8.58 9.72 8.44

Initial temperature (°C) 25 25 25 25

Rate (°C/minute)

- 5

- 5

- 5

- 5

Temperature 2 (°C)

- 90

- 90

- 90

- 90

Rate (°C/minute) 20 20 20 20

End temperature (°C) 168 168 168 400

The atmospheric pressure was 974.7

- 1012 hPa. The experiments were performed under a flow of

nitrogen.

7.3. Analytical method

Preliminary test

Instrument Q50 thermogravimetricanalyzer (TGA) (TA Instruments, New

Castle, DE, USA)

Sample container aluminium; closed with a lid in which a small hole was drilled

Main study

Instrument Q100 differential scanning calorimeter (TA Instruments)

Sample conta iner aluminium; closed (Experiment 2) or closed with a lid in which a

small hole was drilled (Experiment s 1, 3 and 4)

Reference empty sample container

7.4. Interpretation

The melting temperature is defined as the temperature at which the phase transition from solid to

liquid state takes place at normal atmospheric pressure. This temperature ideally corresponds to the

solidification or freezing temperature.

The standard boiling temperature is defined as the temperature at which the vapour pressure of a

liquid is the same as the standard pressure (1013.25 hPa).Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 11

- 7.5. Results

7.5.1. Preliminary test

The TGA

- curve of the test substance is shown in Figure 1

. Starting at 50°C

, the weight of the sample

decreased significantly. At 168°C the sample weight had decreased by 25%. This was most likely due

to the evaporation of water in the test substance.

After the experiment, a yellow/brown solid residue remained in the sample container and on the lid of

the container. (original colour: clear amber liquid). The slight change of the colour indicated some

reaction and/or decomposition of the test substance.

7.5.2. Main study

The DSC

- curve obtained with Experiment 1 is shown in Figure 2

. In the cooling curve, an exothermic

peak between

- 25°C and

- 50°C was observed. The effect was most likely obtained due to

crystallization of water in the test substance. In the heating curve, an endothermic peak between

- 25°C and 25°C was observed with a maximum at

- 4.27°C. The effect was most likely obtained due to

melting of water in the test substance

. S tarting at 75°C a broad endothermic effect with a maximum at

113.28°C was observed most likely obtained due to evaporation/boiling. After the experiment, a light

yellow very viscous almost solid residue, remained in the sample container and on the lid of the

sample container

.

To investigate the evaporation p eak a closed sample container was used in Experiment 2

. Figure 3

shows the DSC

- curve obtained. The temperature program was a duplicate of Experiment 1. M elting

was observed with a maxi mum at

- 4.71°C. The extrapolated onset temperature of the evaporation

peak was 149.41°C. T he endothermic peak shifted to higher t emperatures. It demonstrated that

evaporation , most likely due to water in the test substance, was the reason for the endothermic effect.

After the experiment, a yellow residue remained in the sample container.

Experiment 3 was performed as a duplicate of Experiment 1. Figure 4 shows the DSC

- curve obtained.

Melting was observed with a maximum at

- 3.12°C. The maximum of the evaporation peak was at

113.41°C. After the experiment, a light yellow very viscous almost sol id residue remained in the

sample container and on the lid of the sample container

.

Experiment 4 was performed to investigate any effects after the water had evaporated. Similar results

as with Experiment 1 were obtained. Figure 5 shows the DSC curve. Melting was observed with a

maximum at

- 4.94 °C. The maximum of the evaporation peak was at 112.52°C. A small, broad

endothermic effect was observed from 225°C to 300 °C , most likely due to reaction/decomposition. No

further evaporation/boiling effects were observed. After the experiment, the container was empty . O n

the lid of the container

, a brown residue remained.

The melting temperature was determined as the average peak temperature obtained from

E xperiment 1 (

- 4.27°C), Experiment 2 (

- 4.71°C), Experiment 3 (

- 3.12°C) and E xperiment 4 (

- 4.94°C).

The boiling temperature was determined as the average peak temperature obtained from

E xperiment 1 (113.28°C), Experiment 3 (113.41°C) and E xperiment 4 (112.52°C).

These values are most likely due to water in the test substance (i.e. 24.3%, see chapter 8 )

.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 12

- 7.6. Conclusion

The melting and boiling temperature of Castor Oil, Sulfated, Sodium Salt, 75% were determined using

DSC.

The melting temperature of the test substance was

- 4 °C (269K)

The boiling temperature of the test substance was 113 °C (386K).

The values are most likely due to water in the test substance

.

The atmospheric pressure was 974.7

– 1012 hPa.

7.7. Figures

Figure 1 TGA curve of the test substance. 167.59°C 25.00% Loss

431.33°C 70.00% Loss

20 40 60 80 100 120Weight (%)

0 100 200 300 400 500

Temperature (°C) File: NOTOX2010 13822 62665

Universal V4.7A TA InstrumentsCastor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 13

- Figure 2 DSC curve of the test substance obtained with Experiment 1.

Figure 3 DSC curve of the test substance obtained with Experiment 2. -4.27°C

113.28°C

-150 -100 -50

0 50Heat Flow (mW)

-100 -50 0 50 100 150 200

Temperature (°C) File: NOTOX2010 13829 67430

Exo Up Universal V4.7A TA Instruments

-4.71°C 149.41°C

-30 -20 -10

0 10Heat Flow (mW)

-100 -50 0 50 100 150 200

Temperature (°C) File: NOTOX2010 13829 67587

Exo Up Universal V4.7A TA InstrumentsCastor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 14

- Figure 4 DSC curve of the test substance obta ined with Experiment 3.

Figure 5 DSC curve of the test substance obtained with Experiment 4. -3.12°C

113.41°C

-80 -60 -40 -20

0 20Heat Flow (mW)

-100 -50 0 50 100 150 200

Temperature (°C) File: NOTOX2010 13829 67734

Exo Up Universal V4.7A TA Instruments

112.52°C -4.94°C

-100 -80 -60 -40 -20

0 20Heat Flow (mW)

-100 0 100 200 300 400

Temperature (°C) File: NOTOX2010 13836 45342

Exo Up Universal V4.7A TA InstrumentsCastor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 15

- 8. WATER CONTENT KARL F IS C HER

8.1. Reagents

Hydranal ® Coulomat AK Sigma Aldrich, Steinheim, Germany

Art.nr. 348 20

, Batch SZBC3050V

Hydranal ® Coulomat CG

- K Sigma Aldrich

Art.nr. 34821, Batch SZBB084A

Hydranal ® Water standard 0.1 mg/g = 0.01% H 2 O Sigma

- Aldrich

Art.nr. 34847, Batch SZBA3070

Hydranal ® Water standard 1.0 mg/g = 0.1% H 2 O Sigma

- Aldrich

Art.nr. 34828, Batch SZBD0090

All reagents were of analytical grade, unless specified otherwise.

8.2. Performance of the test

The water content was determined by a Coulometric Karl Fischer titration using a Metrohm 8 31 KF

Coulometer (Metrohm, Herisau, Switzerland).

The system consisted of a reaction vessel which was equipped with a double platinum electrode used

for the instrumental end point indication. In addition to the double platinum electrode, a generator

electrode was also present in the reaction vessel. Prior to titration, the reaction vessel was filled with

100 ml Hydranal Coulomat AK reagent and the generator electrode was filled with one ampoule of

Hydranal Coulomat CG -K reagent. The generator electrode and the reaction vessel were connected

via a porous membrane which facilitated the conduction of electrical current between the two

compartments but prevented the exchange of fluids. The reaction vessel was equipped with a septum

via which liquid samples were introduced using a syringe. Titration was performed by applying a

current to the generator electrode which resulted in the formation of iodine (I 2 ) in the reaction vessel

which was consumed in the Karl Fischer titration process.

The test substance was dissolved in a suitable solvent

. Prior to analysis of the test sample, the system

was first titrated water free. Subsequently the test sample was introduced and titrated.

The results were corrected for the water content in the solvent used for preparation of the test solution

.

Calculations o f t he water content (in %) of the test substance is described in paragraph 8.4.1

. The water content was included in a certificate of analysis

, issued based on these measurements. The

certificate of analysis is shown i n Appendix 1

.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 16

- 8.3. Analytical method

8.3.1. Analytical conditions

Instrument 8 3 1 KF Coulometer (Metrohm, Herisau, Switzerland )

M agnetic stirrer Ti Stand 703 ( Metrohm )

8.3.2. Calibration

The validity of the system was verified using two water standards with a certified water amount of 0.1

and 1 .0 mg/g (Sigma

- Aldrich).

Duplicate samples of each standard were weighed and the water content was determined by titration.

8.3.3. Preparation of solutions

22.2 and 40.2 mg test substance was dissolved in approximately 6 g solvent Hydranal ® Coulomat AK

in a 10 ml vial.

For determination the water content in the solvent, Hydranal ® Coulomat AK was used .

8.4. Interpretation

8.4.1. formulas

Test substance fraction of t he sample solution % *

w w w f

TS M TS

TS 100

+ =

where:

f TS : fraction of test substance in the sample solution [%]

w TS : weight of test substance in the sample solution [g]

w M : weight of solvent in the sample solution [g]

Solvent fraction of the sample solution % *

w w w f

TS M M

M 100

+ =

where:

f M : fraction of solvent in the sample solution [%]

Water content of test substance % *

f f

wc wc

wc

TS M

M ss

TS 100 100      

−

=

where:

wc TS : water content of the test substance [%]

wc SS : water content of the sample solution [%]

wc M : water content of solvent [%]Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 17

- 8.5. Results

Table 1 show the calibration results. The results of the Karl Fischer titrations of the solvent and test

substance are given in Table 2 and Table 3

, respectively

.

Table 1 Calibration of Karl Fischer apparatus

Da te of

analysis Water

standard

[mg/g] Certified

water content

[mg/g] Analysed

water content

[mg/g] Recovery

[%] Mean

recovery

[%]

12

- Oct

- 2013 0.1 0.104 0.106 102 1 1 14

0.119 114

0.119 114

0.119 115

1.0 1.00 1.01 101 10 2

1.0 3 10 3

1 Outlier according to the Dixon’s Q

- test at 95% confidence level.

The results of the 0.1 mg/g standard are somewhat higher than expected (i.e. 114%)

. The recovery of

the 1.0 mg/g standard h owever is 102%. T he concentrations of the sample solutions were in the range

of the 1.0 mg/g standard . T he results were therefore accepted. The system was found to be accurate

for the determination of the water content of the test substance.

Table 2 W ater content of the solvent solution

Date of

analysis W eight solvent

[ g] Analysed

water

[ ppm ] Mean Analysed

water

[ ppm ]

12

- Oct

- 2013 1.631 62.9 71.1

1.545 79.2

Table 3 Water content of the test substance

Date of

analysis W eight test

substance

[ g] W eight solvent

[ g] Analysed

water

[ ppm ] Analysed

water content

[% w/w] Mean water

content

[% w/w]

12

- Oct

- 2013 40.2 5851.0 1731.7 24.3 24.3

22.2 5894.6 982.1 24.3

8.6. Conclusion

The water content of Castor Oil, Sulfated, Sodium Salt, 75% was determined by a Coulometric Karl

Fischer titration.

The water content was 24.3 % w/w (n = 2)

.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 18

- 9. CONTENT SODIUM SULPH ATE

9.1. Guidelines

Food and Agriculture Organization of the United Nations (FAO): Manual on development and use of

FAO and WHO specifications for pesticides, March 2006 revision of the first edition, prepared by the

FAO/WHO joint meeting on pesticide specifications (JMPS), Rome, 2006.

European Commission: Guidance for generating and reporting methods of analysis in support of pre

and post

- registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section

5) of Directive 91/414, SANCO/3030/99 rev. 4 (11/07/00).

9.2. Reagents

Water Tap water purified by a Milli

- Q water pu rification system

(Millipore, Bedford, MA, USA)

Lead(II)nitrate Sigma Aldrich, Steinheim, Germany

Art.nr. 228621, Batch BCBL4721V

Potassium sulphate Sigma Aldrich

Art.nr. 221325, Batch MKBM2228V

Hydroxylamine hydrochloride Sigma Aldrich

Art.nr. 379921, Batch MKBP1350V

Dithizone Sigma Aldrich

Art.nr. 33154, Batch BCBK0249V

Hydrochloric acid 1 M Merck, Darmstadt, Germany

Art.nr.1.09057, Batch HC260857

Sodium hydroxid e 1 M Merck

Art.nr. 1.09137, Batch HC394982

2

- Propanol Merck

Art.nr.1.01040, Batch K44623040.324

All reagents were of analytical grade, unless specified otherwise.

9.3. Reagent solutions

Lead(II)nitrate solution 0.039 M 13.25 g l ead(II)nitrate was dissolved in 1000 ml water

Potassium sulphate solution 0.025 M 4.35 9 g potassium sulphate was dissolved in 1000 ml water

9.4. Performance of the study

A titration method for the quantitative analysis of the available sodium sulphate c ontent supplied by the

sponsor (Cognis No N

- 149B “sulfate (IRAM method)” ) was implemented. Vali dation of the analytical

method was performed for the following parameters.

Specificity

A blank sample was prepared in three

- fold . The analytical method was found to be specific if the

interferences in the blank did not contribute more than 3% to the vol ume titrated for the test substance.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 19

- Precision (repeatability)

The content of sodium sulphate in the test substance was determined by analysing five independent

solutions of the test substance using the analytical method

, expressed in % w/w content sodi um

sulphate . The mean content, the standard deviation and the % RSD of the test samples were

calculated.

A certificate of analysis (see appendix I) was prepared based on the results of the five independent

samples of the test substance.

Accuracy

Five independent accuracy samples were prepared by addition of an amount of potassium sulphate

solution to the test substance. The mixtures were analyzed by the analytical method.

9.5. Analytical method

9.5.1. Analytical conditions

Titrator Titrino 716 DMS (Metrohm, Herisau, Switzerland)

Stirrer 703 TI Stand (Metrohm )

Titration solution Lead(II)nitrate solution

9.5.2. Calibration

The titer of the l ead (II) nitrate solution was determined with a potassium sulphate solution.

4.35 9 g potassium sulphate was accurate ly weighed and dissolved in 1000 ml water (i.e. 0.025 M).

To a beaker of 250 ml, 20 ml potassium sulphate solution was added. Subsequently 70 ml 2

- propanol

and 100 mg hydroxylamine hydrochloride w ere transferred into the beaker. Just enough d ithizone

indicator was added to give the mixture sufficient color after adjusting the pH to 3.1 with hydrochloric

acid 1 M. During titrat ion with l ead(II)nitrate solution, t he pH of the solution was kept between

2.9

- 3.1 with sodium hydroxide 1 M. The endpoint of the titration was reached when a first violet color

was permanent ly observed.

9.5.3. Preparation of solutions

Test samples

To a beaker of 250 ml containing approximately 3000 mg accurately weighed test substance, 30 ml

water was added . The mixture was stirred until dissolution was complete.

Accuracy samples

To a beaker of 250 ml containing approximately 3000 mg accurately weighed test substance, 20 ml

water and 11.6 ml of the 0.025 M potassium sulphate solution w ere added. The mixture was stirred

unti l dissolution was complete.

Blank sample

In a beaker of 250 ml 30 ml water was added.

9.5.4. Sample analysis

To each sample, 70 ml 2

- propanol and 100 mg hydroxylamine hydrochloride were added

. Dithizone

indicator was added just enough to give the solution sufficient color after adjusting the pH to 3.1 with

hydrochloric acid 1 M. The solution was titrated with l ead(II)nitrate solution

. During titration with

l ead(II)nitrate solution, the pH of the solutio n was kept between 2.9

- 3.1 with sodium hydroxide 1 M.

The endpoint of the titration was reached when a first violet color was permanent ly observed.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 20

- 9.6. Interpretation

9.6.1. Formulas

Titer (Lead(II)nitrate solution) t Pb = 

## ( )

bl Pb 

### V V

− C × V

SO4

SO4

where:

t Pb : titer of the Lead(II)nitrate solution (M)

V SO4 : volume potassium sulphate solution (ml)

C SO4 : concentration potassium sulphate solution (M)

V Pb : volume Lead(II)nitrate solution added to sample (ml)

V bl : volume Lead(II)nitrate solution added to b lank (ml)

Content S odium sulphate (C T ) C T = 

### 1000 × w 100% × 142.04 × t × ) (

Pb bl Pb 

### V V

− [% w/w]

where:

t Pb = molarity of lead(II)nitrate ( 0.039 M )

w = sample weight (g)

Analysed sulphate con tent (C A ) Analysed sulphate content of the accuracy samples.

Accuracy 100% C C

N

A

×

where:

C A = analysed sulphate con tent [m mol ]

C N = calculated sulphate content [m mol ]

9.6.2. Precision (repeatability)

The relative standard deviation (% RSD) on the results of the test samples analysed was determined.

Acceptability of this value was based on the Horwitz equation:

C) log 0.5

- (1

R 10 2 RSD % predicted

=

where:

% RSD R = the predicted among

- laboratory RSD

C

= mean content in g/g

For estimation of the repeatability (% RSD

r ) of the titration method the predicted amo ng

- laboratory

RSD is modified to:

0.67 RSD % RSD % predicted R r ×

=

The results of the test samples were accepted if the % RSD for the content sodium sulphate in the

substance was less than the predicted repeatability of the analytical method.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 21

- 9.7. Results

9.7.1. Content Sodium Sulphate

The results of the sodium sulphate content in the test samples are given in Table 5

.

Table 4 Content S odium S ulphate

Code Titration volume

Lead(II)nitrate

[ml] Sample

weight

[ m g] Content

Sodium sulphate

[% w/w]

I 6.692 2996.7 1.22

II 6.912 2996.2 1.26

III 6.910 3004.6 1.26

IV 6.869 2999.0 1.25

V 6.879 3001.1 1.25

Mean content 1.25

Standard deviation 0.0 2

9.7.2. S pecificity

For the blank samples a mean amount of 0.102 ml lead(II)nitrate solution was needed. This volume is

less than 3% (i.e. 1.6%), calculated on the lowest titration volume lead(II)nitrate (see Table 4 )

. Therefore the analytical method was found to be specific for the test substance.

9.7.3. Precision (repeatability)

The relative standard deviation (RSD) of the test samples and the predicted % RSD R and % RSD

r are

given in Table 5

. Since the % RSD for the content sodium sulphate in the test substance was less than

the predicted % RSD

r the results of the test samples were considered precise.

Table 5 Relative standard deviation

% RSD R 3. 9

% RSD

r 2.6

% RSD of the test samples 1. 3Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 22

- 9.7.4. Accuracy

The results of the accuracy samples are given in Table 6

. T he mean recovery result of 97% w as

considered accurate and was therefore accepted

.

Table 6 Accuracy samples

Code Calculated

s piked

s ulphate

[m mol ] Calculated 1

c ontent

sulphate

[m mol ] Calculated total

sulphate content

expected

[m mol ] Analysed

sulphate

content

[m mol ] Recovery

[%]

I 0.290 0.263 0.554 0.536 97

II 0.290 0.264 0.554 0.534 9 6

III 0.290 0.263 0.554 0.539 9 7

IV 0.290 0.263 0.554 0.535 97

V 0.290 0.263 0.553 0.537 9 7

Mean recovery 97

1 based on sample weight and s odium sulphate content determined (i.e. 1.2 5% w/w)

9.8. Conclusion

The sodium sulphate content in Castor Oil, Sulfated, Sodium Salt, 75% was determined to be

1.2 5 ± 0.02 % w/w ( 5n;sx 1n =±

− ).Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 23

- 10. CONTENT SO 3

10.1. Guidelines

Food and Agriculture Organization of the United Nations (FAO): Manual on development and use of

FAO and WHO specifications for pesticides, March 2006 revision of the first edition, prepared by the

FAO/WHO joint meeting on pesticide specifications (JMPS), Rome, 2006.

European Commission: Guidance for generating and reporting methods of analysis in support of pre

and post

- registr ation data requirements for Annex II (part A, Section 4) and Annex III (part A, Section

5) of Directive 91/414, SANCO/3030/99 rev. 4 (11/07/00).

10.2. Reagents

Water Tap water purified by a Milli

- Q water purification system

(Millipore, Bedford, MA, USA)

Benzet honium chloride (Hyamine) Sigma Aldrich, Steinheim, Germany

Art.nr. B8879, Batch SLB C 7904 V

Methylene blue Sigma Aldrich

Art.nr. M9140, Batch SLBF3786V

Sodium dodecyl sulphate (SDS) Sigma Aldrich

Art.nr. L 4390

, Batch 129K01 8 7

Sodium sulphate Merck, Darmstadt, Germany

Art.nr. 1.06649, Batch A0177249 111

Sulphuric acid 96% Merck

Art.nr. 1.00714, Batch K43188714 206

Chloroform VWR International, Leuven, Belgium

Art.nr. 83626.320, Batch 13 Z 4042

All reagents were of analytical grade, unless specified otherwise.

10.3. Reagent solutions

Hyamine solution (0.004 M) 1849 mg hyamine was dissolved in 1000 ml water

Methylene blue solution 60.2 mg methylene blue, 100 g sodium sulphate and 13 ml

sulphuric acid 96% were dissolved to 2000 ml with water.

SDS solution 568.4 mg S odium Dodecyl Sulphate was dissolved to 100.0 ml

with water.

10.4. Performance of the study

A titration method for the quantitative analysis of the SO 3 c ontent supplied by the sponsor (CTMC

- 89A

” Determination of SO 3 ” ) was implemented. Valid ation of the analytical method was performed for the

following parameters.

Precision (repeatability)

The SO 3 content in the test substance was determined by analysing five independent solutions of the

test substance using the analyt ical method, expressed in % w/w SO 3 content . The mean content, the

standard deviation and the % RSD of the test samples were calculated.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 24

- A certificate of analysis (see appendix I) was prepared based on the results of the five independent

samples of the test substance.

Accuracy

Two independent accuracy samples were prepared by addition of an amount of SDS solution to the

test substance. The mixtures were analyzed by the analytical method.

10.5. Analytical method

10.5.1. Analytical conditions

Titrator Titrino 716 DMS (Metrohm, Herisau, Switzerland)

Stirrer 703 TI Stand (Metrohm)

Titration solution Hyamine solution

10.5.2. Preparation of solutions

Test samples

An accurately weigh ed amount of approximately 100 mg test substance was quantitatively transferred

with approximately 50 m l wat er, in to a conical flask of 250 ml.

Accuracy samples

An accurately weigh ed amount of approximately 100 mg test substance was quantitatively transferred

with approximately 50 m l water, in to a conical flask of 250 ml. The solution was spiked with 2.0 ml

SDS solution.

10.5.3. Sample analysis

To each sample, 75 ml chloroform and 30 ml methylene blue solution were added . Stirring of the

mixture was st arted . An aliquot hyamine solution

, 3 ml less than the calculated end volume

, was

added. T he mixing was stopped to allow the two layers to separate. The addition of the hyamine

solution was repeated until both layers ha d the same intensity of the blue color ation . Then t he

endpoint of titration wa s reached

.

10.6. Interpretation

10.6.1. Formulas

% SO 3 content (C T ) C T = 

### 1000 × w 100% × 80.07 × t × V [%w/w]

where:

V

= ml hyamine solution

t

= tite r of hyamine solution ( 0.004 M )

w = sample weight (g)

Analysed concentration (C A ) Analysed concentration of the accuracy samples.

Accuracy 100% C C

N

A

×

where:

C A = analysed concentration SO 3 [m mol ]

C N = calculated concentration SO 3 [m mol ]Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 25

- 10.6.2. Precision (repeatability)

The relative standard deviation (% RSD) on the results of the test samples analysed was determined.

Acceptability of this value was based on the Horwitz equation:

C) log0.5

- (1

R 10

2 RSD % predicted

=

where:

% RSD R = the predicted among

- laboratory RSD

C = mean content in g/g

For estimation of the repeatability (% RSD

r ) of the titration method the predicted among

- laboratory

RSD is modified to:

0.67 RSD % R SD % predicted R r ×

=

The results of the test samples were accepted if the % RSD for the analysis of the active ingredient in

the test substance was less than the predicted repeatability of the analytical method.

10.7. Results

10.7.1. SO 3 content

The results of the test samples are given in Table 7

.

Table 7 SO 3 content

Code Titration volume

hyamine solution

[ml] Sample

weight

[ m g] SO 3 content

[% w/w]

I 17.523 104.5 5.54

II 18.134 107.5 5.58

III 16.988 100.5 5.59

IV 16.126 95.7 5.57

V 17.441 102.2 5.64

Mean content 5.58

Standard deviation 0.04

10.7.2. Precision (repeatability)

The relative standard deviation (RSD) of the test samples and the predicted % RSD R and % RSD

r are

given in Table 8

. Since the % RSD for the analysis of the active ingredient in the test substance was

less than the predicted % RSD

r the results of the test samples were considered precise.

Table 8 Relative standard deviation

% RSD R 3. 1

% RSD

r 2.1

% RSD of the test samples 0.65Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 26

- 10.7.3. Accuracy

The results of the accuracy samples are given in Table 6

. T he mean recovery result of 104% was

con sidered accurate and was therefore accepted.

Table 9 Accuracy samples

Code Spiked

SO 3 content

[m mol ] Sample

contribution

SO 3 content

[m mol ] Total

SO 3 content

[m mol ] Analysed

SO 3 content

[m mol ] Recovery

[%]

I 0.0394 0.0717 0.111 0.119 107

II 0.0394 0.0710 0.110 0.112 102

Mean recovery 104

10.8. Conclusion

The SO 3 content in Castor Oil, Sulfated, Sodium Salt, 75% was determined to be

5.58 ± 0.04 % w/w ( 5n;sx 1n =±

− ).

11. PURITY / COMPOSITION

The purity / composition of Castor Oil, Sulfated, Sodium Salt, 75% was calculated as 100 % minus the

water a nd sodium sulphate content

.

Using a water content of 24.3 % and a sodium sulphate content of 1.2 5%

, purity / composition is

calculated to be 74.4 %.Castor Oil, Sulfated, Sodium Salt, 75% Project 503335

Final report

- Page 27

- Appendix 1 Certificate of analysis