FINAL REPORT

Study Title

DETERMINATION OF PHYSICO-CHEMICAL PROPERTIES OF 

FRET 09-0536

 Validation of an analytical method

 Melting temperature

 Vapour pressure

 Water solubility

 Partition coefficient

Author

I. Lammers, PhD. 

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s-Hertogenbosch

The Netherlands

 Laboratory Project Identification

Project 504371

Substance 205292/A

- Page 1 of 34 -
FRET 09-0536 Project 504371

Final report - Page 2 - 

1. CONTENTS

1. CONTENTS ......................................................................................................................................2

2. STATEMENT OF GLP COMPLIANCE.............................................................................................4

3. QUALITY ASSURANCE STATEMENT ............................................................................................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. VALIDATION OF AN ANALYTICAL METHOD.................................................................................9

7.1. Guidelines...............................................................................................................................9

7.2. Reagents ................................................................................................................................9

7.3. Performance of the study .......................................................................................................9

7.4. Analytical method ................................................................................................................ 10

7.5. Formulas.............................................................................................................................. 11

7.6. Results................................................................................................................................. 12

7.7. Conclusion ........................................................................................................................... 13

7.8. Figures................................................................................................................................. 14

8. MELTING TEMPERATURE........................................................................................................... 15

8.1. Guidelines............................................................................................................................ 15

8.2. Performance of the test ....................................................................................................... 15

8.3. Analytical method ................................................................................................................ 16

8.4. Interpretation........................................................................................................................ 16

8.5. Results................................................................................................................................. 16

8.6. Conclusion ........................................................................................................................... 16

8.7. Figures................................................................................................................................. 17

9. VAPOUR PRESSURE................................................................................................................... 18

9.1. Guidelines............................................................................................................................ 18

9.2. Performance of the test ....................................................................................................... 18

9.3. Analytical method ................................................................................................................ 18

9.4. Reference substances......................................................................................................... 19

9.5. Interpretation........................................................................................................................ 20

9.6. Results................................................................................................................................. 21

9.7. Conclusion ........................................................................................................................... 21

9.8. Figures................................................................................................................................. 22

10. WATER SOLUBILITY.................................................................................................................... 23

10.1. Guidelines............................................................................................................................ 23

10.2. Reagents ............................................................................................................................. 23

10.3. Performance of the study .................................................................................................... 23

10.4. Analytical method ................................................................................................................ 25

10.5. Interpretation........................................................................................................................ 26

10.6. Results................................................................................................................................. 26

11. PARTITION COEFFICIENT ........................................................................................................... 28

11.1. Guidelines............................................................................................................................ 28

11.2. Reagents ............................................................................................................................. 28

11.3. Performance of the study .................................................................................................... 28

11.4. Analytical method ................................................................................................................ 29

11.5. Interpretation........................................................................................................................ 30

11.6. Results................................................................................................................................. 30

11.7. Conclusion ........................................................................................................................... 31

11.8. Figures................................................................................................................................. 32
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TABLES

Table 1 Statistical parameters of the calibration curve .................................................................. 12

Table 2 Accuracy samples ............................................................................................................. 12

Table 3 Stability of the analytical system and end solutions.......................................................... 13

Table 4 Results of the isothermal TGA analysis ............................................................................ 21

Table 5 Vapour pressure of the test substance ............................................................................. 21

Table 6 Main study - water solubility of the test substance ........................................................... 27

Table 7 Partition coefficient of the test substance ......................................................................... 31

FIGURES

Figure 1 GC-FID chromatogram of a 20.0 mg/l test substance solution [res. id. 1503].................. 14

Figure 2 GC-FID chromatogram of the blank accuracy sample [res. id. 1609]............................... 14

Figure 3 Regression line: response of the test substance as a function of concentration 

[cal. curve id. 1493]........................................................................................................... 14

Figure 4 TGA curve of the test substance....................................................................................... 17

Figure 5 DSC curve of the test substance obtained with the heating of Experiment 1................... 17

Figure 6 Weight loss curve of the test substance. .......................................................................... 22

Figure 7 Plot of log PT of the test substance versus 1/T................................................................. 22

Figure 8 HPLC chromatograms of the 750 mg/l test solution [top; res. id. 1286] and 

corresponding blank [bottom; res. id. 1285]...................................................................... 32

Figure 9 The regression line of the reference substances: log k’ versus log Pow. .......................... 32

APPENDICES

Appendix 1 Certificate of analysis ........................................................................................................ 33

Appendix 2 Substance Identity Information Document for FRET 09-0536 (multi-constituent)............ 34


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4. SUMMARY

The results of the physico-chemical properties of the test substance are given below. 

Parameter Guideline(s) Result Comment

Analytical method SANCO 3029 successfully validated

Melting temperature EC A.1 

OECD 102

OPPTS 830.7200

< -82°C (< 191K)

Vapour pressure EC A.4

OECD 104

OPPTS 830.7950

20°C 1.1 Pa = 

8.4 × 10-3 mm Hg

25°C 1.8 Pa = 

1.4 × 10-2 mm Hg

isothermal TGA 

effusion method

Water solubility EC A.6

OECD 105

OPPTS 830.7840

0.0134 g/l at 20°C by the slowstirring flask method

Partition coefficient EC A.8

OECD 117

OPPTS 830.7570

log Pow of 3.9 - 4.8 for 

twelve overlapping 

components that form 

95% of the total peak area

HPLC method
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5. INTRODUCTION

5.1. Preface

Sponsor International Flavors & Fragrances Inc.

800 Rose Lane

Union Beach, New Jersey 07735

U.S.A.

Study Monitor Vijyeta Kapur

Senior Regulatory Chemical Control Specialist

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s-Hertogenbosch

The Netherlands

Study Director I. Lammers, PhD.

Study Plan Start : 16 January 2014

Completion : 19 June 2014

5.2. Aim of the study

The aim of the study was to determine the following physico-chemical properties for FRET 09-0536:

 Validation of an analytical method

 Melting temperature

 Vapour pressure

 Water solubility

 Partition coefficient

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 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 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.
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6. MATERIALS

6.1. Test substance

6.1.1. Test substance information

Identification FRET 09-0536

Structure

H

H

O

+

H

H O

Molecular formula C13H20O

Molecular weight 192.15

CAS Number 1464989-16-9

Description Clear colourless liquid

Batch RDIA5595

Purity/Composition 89.6%

Test substance storage In refrigerator (2-8°C) in the dark under nitrogen

Stable under storage conditions until 01 November 2014 (expiry date) 

6.1.2. Study specific test substance information

Purity/composition correction factor 

required

No

Volatile Yes, vapour pressure: 0.024 Pa at 298K

Reactivity Reactive to oxygen

Test substance handling. Flush container with nitrogen after handling

6.2. Electronic data capture

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

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

- Empower version 7.00 (Waters, Milford, MA, USA)

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

Water solubility test (see Paragraph 10.6.5): The maximum difference between 24- and 72-hours 

measurements is larger than the criterion ≤ 15% (i.e. 18%). The deviation above 15% is small, thereby 

limiting the effect on the study. Prolongation of the stirring time was not desirable considering reactivity 

of the test substance with oxygen as indicated by the sponsor. The study integrity was not adversely 

affected by the deviation.

6.3.2. List of standard operating procedures deviations

There were no deviations from standard operating procedures (SOPs).
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7. VALIDATION OF AN ANALYTICAL METHOD

7.1. Guidelines

The study was based on the following guideline:

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

Pre-registration Data Requirements 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. Reagents

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA)

Cyclohexane Merck, Darmstadt, Germany

Acetone Merck

All reagents were of analytical grade, unless specified otherwise.

7.3. Performance of the study

A gas chromatographic method with flame ionization detection (GC-FID) for quantitative analysis of 

the test substance in water was developed. Validation of the analytical method was performed for the

following parameters:

Specificity

A test substance solution and blank accuracy sample were analysed by single injection. The analytical 

method was found to be specific if the blank chromatogram showed no response for the test 

substance or a response of < 30% of the limit of quantification. 

Calibration curve

Calibration solutions were analysed in duplicate. The response of the calibration solutions was 

correlated with concentration using regression analysis with a 1/concentration2 weighting factor. A 

calibration curve with a coefficient of correlation (r) of > 0.99 and back calculated accuracies of the 

calibration solutions in the range 85-115% was accepted. 

Accuracy and repeatability

Accuracy samples were analysed by single injection into the analytical system. The analytical method 

was considered applicable for the determination of the test substance if the mean accuracy was in the 

range 70-110% and the coefficient of variation was ≤ 20%.

Limit of quantification

The limit of quantification (LOQ) is defined as the lowest concentration level at which an accuracy in 

the range 70-110% and a repeatability of ≤ 20% is demonstrated. The LOQ was obtained from the

data of the accuracy- and repeatability test.

Stability of the analytical system and end solutions

Calibration solutions were injected throughout the validation sequence including the beginning and 

end. The analytical system and/or end solutions were found to be stable if the coefficient of variation 

on the responses of the solutions was ≤ 20%.

Stability of stock solutions

Stock solutions of the test substance were stored at room temperature for at least 12 hours. Additional 

calibration solutions were prepared and analysed by single injection. The stock solutions were found to 

be stable if the coefficient of variation on the response factors of the calibration solutions prepared with 

fresh and stored stock solutions was ≤ 10%.
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7.4. Analytical method

7.4.1. Analytical conditions

Instrument Gaschromatograph 6890N 

(Agilent Technologies, Palo Alto, CA, USA) 

Injector CombiPal (CTC analytics, Zwingen, Switzerland)

Detector Flame ionisation detector (Agilent Technologies)

Column DB-5ms; 30 m × 250 μm i.d., df = 1.00 μm

(J&W Scientific, Folsom, CA, USA)

Carrier gas Helium 

Carrier gas flow 1 ml/min (constant flow)

Injection Programmed temperature vaporizer (PTV)

Splitless time 60 sec

Split flow 50 ml/min

Injection volume 1 μl

PTV temperature program

Initial temperature 70°C 

Initial time 0.1 min

Rate 720°C/min

Final temperature 250°C

Hold time 5 min

Oven temperature program

Initial temperature 70°C 

Initial time 1 min

Rate 1 50°C/min

Temperature 1 210°C

Hold time 8 min

Rate 2 50°C/min

Final temperature 240°C

Hold time 2.6 min

FID detection 

Temperature 250°C

Hydrogen 40 ml/min

Air 450 ml/min 

Nitrogen (make-up) 50 ml/min 

7.4.2. Preparation of solutions

Stock and spiking solutions 

Stock solutions of the test substance were prepared in cyclohexane at concentrations of 

2000 - 2500 mg/l. 

Spiking solutions of the test substance were prepared in acetone at concentrations of 25 - 5000 mg/l.

Calibration solutions

Five calibration solutions in the concentration range of 0.2 – 20 mg/l were prepared from two stock

solutions. The end solution of the calibration solutions was cyclohexane.
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Accuracy samples 

3 ml water was spiked with the test substance at a target concentration of 0.5 or 100 mg/l. The

accuracy samples were extracted in 1:1 (v:v) ratio with cyclohexane. The shaking time was 

30 seconds. If necessary, the samples were further diluted with cyclohexane to obtain concentrations 

within the calibration range. 

The blank accuracy sample was prepared and treated similarly to the accuracy samples.

7.5. Formulas

Response (R) Peak area of the test substance [units]

Response factor (Rf)

N

f C

R R =

where:

CN = nominal concentration [mg/l]

Calibration curve R = a CN + b

where:

a = slope [units × l/mg]

b = intercept [units]

Analysed concentration (CA) d [mg/l]

a

(R b) CA × − =

where:

d = dilution factor

Accuracy 100 [%] C

C

N

A ×
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7.6. Results

7.6.1. Specificity

Chromatograms of a test substance solution and the blank accuracy sample are shown in Figure 1

and Figure 2, respectively. 

Several small peaks and one large peak were observed in the chromatogram of the test substance 

solution. It was assumed that the large peak derives from the major component of the test substance, 

whereas the small peaks derive from impurities. The peak area of the major component was used as 

response in the calculations. 

The chromatogram of the blank sample showed no peak at the retention time of the test substance. 

Since no interferences were detected, the specificity requirements were met and the analytical method 

was found to be specific for the test substance.

7.6.2. Calibration curve

The calibration line was constructed using all data points. Figure 3 illustrates the calibration curve and

Table 1 shows the statistical parameters. There was a linear relationship between response and test 

substance concentration in the range of 0.200 – 20.0 mg/l (in end solution). Since the coefficient of 

correlation (r) was > 0.99 and the back calculated accuracies of the data points were in the range 

85-115% the calibration line was accepted.

Table 1 Statistical parameters of the calibration curve

Slope 5.10

Intercept -1.46 × 10-2

Weighting factor 1/concentration2

r 0.998

7.6.3. Accuracy and repeatability 

The results of the accuracy samples are given in Table 2. Since the mean accuracy at each 

concentration level fell in the criterion 70-110% and the coefficient of variation was ≤ 20% the 

analytical method was accepted for the analysis of the test substance in water in the target

concentration range of 0.5 - 100 mg/l.

Table 2 Accuracy samples

Target 

concentration

[mg/l]

Nominal

concentration

[mg/l]

Analysed 

concentration

[mg/l]

Accuracy 

[%]

Mean 

accuracy 

[%]

Coefficient of 

variation 

[%]

0.5 0.500 0.456 91 94 2.9

0.500 0.459 92

0.500 0.473 95

0.500 0.480 96

0.500 0.488 98

100 100 96.8 97 103 4.2

100 99.2 99

100 106 106

100 107 107

100 104 104
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7.6.4. Limit of quantification

The limit of quantification (LOQ) was assessed at 0.5 mg/l in water. 

7.6.5. Stability of the analytical system and end solutions

The results of the stability of the analytical system and end solutions are given in Table 3. Since the 

coefficient of variation at all concentration levels was ≤ 20% the analytical system and end solutions

were stable over at least a 13.8 hour time interval.

Table 3 Stability of the analytical system and end solutions

Nominal concentration

[mg/l]

Elapsed time

[hours]

Coefficient of variation

[%]

0.200 13.8 5.7 (n=6)

0.675 13.8 3.7 (n=6)

20.0 16.3 1.9 (n=8)

7.6.6. Stability of stock solutions

The coefficient of variation on the response factors of the calibration solutions prepared with fresh and 

stored stock solutions was 1.7%. Since the value was ≤ 10% the stock solutions were stable when 

stored at room temperature for at least 6 days.

7.7. Conclusion

The analytical method was validated for the following parameters:

Specificity specific

Calibration curve r = 0.998

Accuracy 94 and 103%

Repeatability 2.9 and 4.2%

Limit of quantification 0.5 mg/l

Stability analytical system and end solutions stable

Stability stock solutions stable
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7.8. Figures

Figure 1 GC-FID chromatogram of a 20.0 mg/l test substance solution [res. id. 1503].

Figure 2 GC-FID chromatogram of the blank accuracy sample [res. id. 1609].

Figure 3 Regression line: response of the test substance as a function of concentration 

[cal. curve id. 1493].

TS205292 - 10.169

pA

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

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70.00

75.00

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85.00

90.00

95.00

100.00

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

pA

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

65.00

70.00

75.00

80.00

85.00

90.00

95.00

100.00

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Area -20.00

-10.00

0.00

10.00

20.00

30.00

40.00

50.00

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Amount

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00
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8. MELTING TEMPERATURE

8.1. Guidelines

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.1: “Melting/Freezing Temperature”, Official Journal of the European 

Union no. L142, May 31, 2008.

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

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

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

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

8.2. Performance of the test

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

(DSC). This technique records the difference in heat flow of two crucibles, 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, it is 

indicated by a deviation from the base line of the heat-flow record. Because melting is a process in 

which heat is consumed, for these processes the deviation from the base line is in the endothermic 

direction. The melting temperature (if any) of the test substance is evaluated from the DSC curve as 

the extrapolated onset temperature of the endothermic melting peak. 

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

analyzer (TGA). 

8.2.1. Preliminary test

6.28 mg test substance was heated at a rate of 20°C/minute from 25°C to 300°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 substance was determined.

8.2.2. Main study

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

Experiment 1

Sample amount (mg) 6.87

Initial temperature (°C) 25

Rate (°C/minute) -5

Temperature 2 (°C) -90

Rate (°C/minute) 20

End temperature (°C) 25

The experiments were performed under a flow of nitrogen and at atmospheric pressure.

Since no crystallization or melting of the test substance was observed, an additional test was 

conducted in which a subsample of the compound was placed overnight in a freezer at -20°C and 

-80°C. The consistency of the test substance was determined after the experiment.
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8.3. Analytical method

Preliminary test

Instrument Q50 thermogravimetric analyzer (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 container aluminium; closed with a lid in which a small hole was drilled

Reference empty sample container

8.4. 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. 

8.5. Results

8.5.1. Preliminary test

The TGA-curve of the test substance is shown in Figure 4. From 200°C upwards the weight of the 

sample decreased significantly. At 275°C the sample weight had decreased by 25%. 

After the experiment a colourless residue remained in the sample container (original: colourless). 

8.5.2. Main study

The DSC-curve obtained with Experiment 1 is shown in Figure 5.

During cooling no effects were observed which were due to crystallization of the test substance 

(results are archived in the raw data). 

During heating no effects were observed which were due to melting of the test substance.

After the experiment, a colourless residue remained in the sample container, the test substance

appeared unchanged. 

To further investigate the melting temperature of the test substance, two samples of the test substance 

were stored overnight at -22.2 ± 0.4°C and -82.0 ± 1.9°C respectively. It was observed after storage 

that the test substance was still a (viscous to highly viscous) liquid in both experiments. According to 

this it was concluded that the melting temperature of the test substance is < -82°C (< 191K). 

8.6. Conclusion

The melting temperature of FRET 09-0536 was determined using DSC and a storage experiment in 

the freezer. 

The melting temperature of the test substance was < -82°C (< 191K). 
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8.7. Figures

Figure 4 TGA curve of the test substance.

Figure 5 DSC curve of the test substance obtained with the heating of Experiment 1.

274.65°C 25.00% Loss

300.16°C 70.00% Loss

20

40

60

80

100

120

Weight (%)

0 50 100 150 200 250 300 350

Temperature (°C)

 File: NOTOX2010 13922 15315

Universal V4.7A TA Instruments

-6

-4

-2

0

2

Heat Flow (mW)

-100 -80 -60 -40 -20 0 20 40

Temperature (°C)

 File: NOTOX2010 13922 15581

Exo Up Universal V4.7A TA Instruments
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9. VAPOUR PRESSURE

9.1. Guidelines

European Community (EC), EC no. 761/2009, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.4: “Vapour Pressure", Official Journal of the European Union no. 

L220, August 24, 2009.

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

of Chemicals no.104: "Vapour Pressure", March 23, 2006.

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

OPPTS 830.7950: "Vapor pressure", August 1996.

9.2. Performance of the test

The vapour pressure of the test substance (PT) was determined by the isothermal thermogravimetric 

effusion method. The method is validated in the range 10-8 - 103 Pa using a set of five reference 

substances with known vapour pressures (see paragraph 9.4). The validity of the method was verified 

maximum one month before this study using hexachlorobenzene as reference control substance. The 

logarithm of the evaporation rate at 20°C (log vT, 20) deviated < 10% from the average value obtained 

during the validation test. From this, it was possible to apply the constants obtained with the validation 

test for the determination of the vapour pressure of the test substance. 

An amount of 6.55 or 7.12 mg of the test substance was applied to the surface of a roughened glass 

plate as a homogeneous layer.

The following temperature program was consequently applied. 

Start temperature 30°C

Isothermal intervals 10 minutes

Increment steps 10°C

End temperature 100°C

The weight loss of the test substance was measured continuously as a function of time at defined 

isothermal temperatures. The evaporation rate of the test substance (vT) was calculated from the 

weight loss of the compound. The PT values at the defined isothermal temperatures were determined 

using the vapour pressure equation with the constants c and d specific for the experimental 

arrangement. Linear regression analysis of the log PT values and 1/T was performed using a least 

squares method. The PT values of the test substance at 20°C and 25°C were determined using the 

vapour pressure regression curve.

9.3. Analytical method

Instrument Q50 thermogravimetric analyzer (TGA) (TA Instruments, New 

Castle, DE, USA)

Surface of the glass plate 0.50 cm2

The experiments were performed under a flow of nitrogen and at atmospheric pressure.
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9.4. Reference substances

Validation of the method is performed at least once every year (project 504840). 

Each reference substance was measured in duplicate by TGA using a temperature program that was 

specific for the substance. Plots of log vT obtained at elevated temperatures and 1/T were inter- or 

extrapolated to determine the log vT values at 20°C (log vT,20). The log vT,20 values were plotted against 

the logarithm of the vapour pressure at 20°C in Pascal units (log PT, 20). Linear regression analysis 

using the least squares method yielded an equation of log PT,20 = 1.14 × log vT,20 + 4.61. The 

coefficient of correlation (r) was > 0.99. The constants c and d specific for the experimental 

arrangement were 1.14 and 4.61, respectively.

Reference PT values 

20°C

Corresponding PT values 

25°C

Literature

Reference substance [Pa] [mm Hg] [Pa] [mm Hg]

Benzo(ghi)perylene 1.33 × 10-8 1.00 × 10-10 2.51 × 10-8 # 1.88 × 10-10 # 1

Chrysene 8.40 × 10-7 6.30 × 10-9 1.31 × 10-6 # 9.80 × 10-9 # 2

Hexachlorobenzene 1.47 × 10-3 1.10 × 10-5 9.59 × 10-3 # 7.20 × 10-5 # 1

Naphthalene 7.56 × 100 5.67 × 10-2 1.15 × 101 8.61 × 10-2 3

Water 2.34 × 103 1.75 × 101 3.17 × 103 2.38 × 101 3

# Determined by the vapour pressure regression curve of the validation study. No literature value was available.

1. Verschueren, K., Handbook of environmental data on organic chemicals, 4th edition, Wiley Chemistry

2. Borges, H.T., Formal toxicity summary for Chrysene, The Risk Assessment Information System (RAIS), 1994

3. Lide, D.R., CRC Handbook of Chemistry and Physics, 79th edition, CRC Press LLC., Boca Raton, FL, USA, 1998

-10

-8

-6

-4

-2

0

2

4

-14 -12 -10 -8 -6 -4 -2 0

log PT,20

log vT,20
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9.5. Interpretation

9.5.1. Specifications

The vapour pressure is a function of the temperature and is specified in Pascal (Pa) or in mm Hg. 

9.5.2. Formulas

Evaporation rate (νT) [g/cm /h] F t

m 2

T ×

∆

ν =

where:

∆m = weight loss of the test substance [g]

F = surface of the sample plate [cm2

]

t = elapsed time for the weight loss [h]

Vapour pressure equation logPT = c logνT + d

where:

c = constant specific for the experimental arrangement

d = constant specific for the experimental arrangement

Vapour pressure regression curve log PT = a 1/T + b

where:

T = temperature [K]

a = slope [K]

b = intercept
FRET 09-0536 Project 504371

Final report - Page 21 - 

9.6. Results

A representative weight loss curve of the test substance is shown in Figure 6. The results of the 

isothermal TGA analysis for the test substance and the vapour pressure at 20°C and 25°C are given in

Table 4 and Table 5.

The plot of the log PT of the test substance as function of the reciprocal temperatures is shown in 

Figure 7. The equation of the curve was: log PT = -3773 × 1/T + 12.92 (r = 0.9998, n = 8). 

Table 4 Results of the isothermal TGA analysis

Temperature

[°C]

Weight loss

[μg/min]

νT 

[g/cm2/h]

log νT log PT PT

[Pa]

50 9.390 1.12 × 10-3 -2.95 1.23 1.7 × 101

9.914 1.18 × 10-3 -2.93 1.26 1.8 × 101

60 19.43 2.32 × 10-3 -2.63 1.59 3.9 × 101

19.48 2.33 × 10-3 -2.63 1.60 3.9 × 101

70 37.88 4.52 × 10-3 -2.34 1.93 8.4 × 101

38.17 4.56 × 10-3 -2.34 1.93 8.5 × 101

80 70.80 8.45 × 10-3 -2.07 2.24 1.7 × 102

70.92 8.47 × 10-3 -2.07 2.24 1.7 × 102

Table 5 Vapour pressure of the test substance

Temperature

[°C]

log PT PT

[Pa]

PT

[mm Hg]

20 0.05 1.1 8.4 × 10-3

25 0.27 1.8 1.4 × 10-2

9.7. Conclusion

The isothermal TGA effusion method was applied for the determination of the vapour pressure of 

FRET 09-0536. 

The vapour pressure of the test substance at 20°C (293K) and 25°C (298K) was:

20°C 25°C

[Pa] [mm Hg] [Pa] [mm Hg]

Test substance 1.1 8.4 × 10-3 1.8 1.4 × 10-2
FRET 09-0536 Project 504371

Final report - Page 22 -

9.8. Figures

Figure 6 Weight loss curve of the test substance.

Figure 7 Plot of log PT of the test substance versus 1/T.

-9.390μg/min

-19.43μg/min

-37.88μg/min

-70.80μg/min

80.00°C

70.00°C

60.00°C

50.00°C

5500

6000

6500

7000

7500

Weight (μg)

20 30 40 50 60 70

Time (min)

 File: NOTOX2010 13923 81320

Universal V4.7A TA Instruments

0

0.5

1

1.5

2

2.5

0.0028 0.00285 0.0029 0.00295 0.003 0.00305 0.0031 0.00315

log PT

1/T [K-1]
FRET 09-0536 Project 504371

Final report - Page 23 -

10. WATER SOLUBILITY

10.1. Guidelines

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.6: “Water Solubility”, Official Journal of the European Union no. 

L142, May 31, 2008.

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

of Chemicals no. 105: "Water Solubility", July 27, 1995.

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

OPPTS 830.7840: "Water Solubility: Column Elution Method; Shake Flask Method", March 1998.

10.2. Reagents

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA)

Double distilled water Waldeck, Münster-Roxel, Germany

Cyclohexane Merck, Darmstadt, Germany

Acetone Merck

Sodium chloride (NaCl) Merck

All reagents were of analytical grade, unless specified otherwise. 

10.3. Performance of the study

Two methods are available for the determination of the water solubility of a test substance: 

1. Column elution method 

The method is based on the elution of the test substance with water from a column which is charged 

with an inert support material, such as bare silica particles, coated with an excess of the test 

substance. It can be applied to test substances with a water solubility of < 10-2 g/l.

2. Flask method 

The mass concentration of the test substance in aqueous solutions saturated with the test substance 

will be determined after stirring or shaking at a specific temperature for a specific period of time. The 

method can be used for test substances with a water solubility of ≥ 10-2 g/l.

A preliminary test was carried out as a range-finding test prior to performance of the column elution 

method or flask method.

Preliminary test 1

Test substance was stirred with double distilled water in a glass vessel at a nominal concentration of 

334 mg/l at 20.1 ± 0.3°C for 19 hours (1 day). After stirring, the water phase was centrifuged in 

polypropylene tubes (three times at 16421 g), treated and analysed. 

Preliminary test 2

A second preliminary test was performed since it was suspected that the test substance reacted with 

the polypropylene tubes during the centrifugation. This test was carried out using both polypropylene 

and glass tubes.
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Final report - Page 24 - 

Test substance was stirred with double distilled water in a glass vessel at a nominal concentration of 

325 mg/l at 20.1 ± 0.4°C for 45 hours (2 days). After stirring, the water phase was centrifuged in 

polypropylene tubes (twice at 3500 g followed by once at 16421 g) as well as in glass tubes (twice at 

35000 g), treated and analysed. 

In the preliminary tests the water solubility of the test substance was < 10-2 g/l. Therefore, the column 

elution method was chosen for the main study.

Main study 1 – column elution method

A solution with an absolute test substance quantity of 74.3 mg was prepared in acetone. The solution 

was mixed with 5 g LiChroprep Si 60 25-40 μm column material (Merck, Darmstadt, Germany). 

Acetone was completely evaporated at 35°C using a rotary evaporator. The loaded carrier material 

was stored overnight at room temperature. 

A 150 mm × 4.6 mm i.d. column was filled with the carrier material and 0.5 μm frits were used to

enclose the column. Double distilled water was pumped through the column until it was completely 

filled. The system was allowed to equilibrate for two hours. 

After the equilibration period, the column was eluted overnight with double distilled water at a flow rate 

of 0.2 ml/hour. The following day the flow was increased to 24 ml/hour. Ten consecutive samples of 2 

ml were taken. The flow was halved to 12 ml/hour and ten consecutive samples of 2 ml were taken. 

Analysis was performed on subsamples of 750 μl. The samples were extracted in a 1:1 (v:v) ratio with 

cyclohexane. The shaking time was 30 seconds. The organic layer was analysed.

The pH was measured from the water fractions that were collected for the determination of the flow 

rates. Each fraction was diluted in a 9:1 (v:v) ratio with 0.1 M sodium chloride in order to obtain a high 

electrolyte concentration for accurate pH measurement.

The eluates were checked for the presence of undissolved particles using a turbidimeter. 

The experiment was also performed using a column filled with blank carrier material. The procedure 

was identical to the procedure used for the column loaded with test substance. At each flow rate, two 

consecutive samples were taken for analyses.

The column elution method was performed at 19.9°C ± 0.2°C.

Since no test substance was detected in the fractions collected from the column, the column material 

was analyzed to demonstrate that the column still contained test substance after elution with water. A 

volume of 5 ml cyclohexane was added to an amount of the loaded carrier material (column material 

from the column after performance of the study and the column material used to prepare the columns).

The solution was ultrasonicated for 5 minutes, centrifuged and thereafter diluted by a factor 10 with 

cyclohexane.

The analysis of the column material indicated that the column contained test substance at very low 

loading at the start of the study. This was probably caused by evaporation of the test substance during 

evaporation of acetone using the rotary evaporator. Therefore, a second column elution study was 

performed with test substance dry mixed with the carrier material.

Main study 2 – column elution method

An absolute test substance quantity of 254.7 mg was mixed with 5 g LiChroprep Si 60 25-40 μm

column material (Merck, Darmstadt, Germany). The mixture was homogenised by rotation for 7 days 

at room temperature.
FRET 09-0536 Project 504371

Final report - Page 25 -

A 150 mm × 4.6 mm i.d. column was filled with the carrier material and 0.5 μm frits were used to

enclose the column. Double distilled water was pumped through the column until it was completely 

filled. The eluate was turbid. The same was observed for a second column. Analyses of the turbid 

eluate confirmed that a large amount of test substance eluted from the column. This indicated that the 

column elution method was not applicable for the determination of the water solubility of the test 

substance. Therefore, an additional water solubility study was performed based on the shake flask 

method in which the flasks were slowly stirred.

Main study 3 – slow-stirring flask method

Three glass vessels were filled with 200 ml double distilled water. To exclude oxygen, nitrogen gas 

was purged through the solution for 5 minutes. 10 μl of the pure test substance was spiked on top of 

the solutions. Nitrogen gas was purged on top of the solution for 1 minutes. The vessels were closed 

and placed on a magnetic stirring device at 19.8 ± 0.3°C where the content of each vessel was stirred 

for 24, 48 or 72 hours at 100 rpm. As the blank, 200 ml double distilled water was stirred for 72 hours. 

After the stirring period, a subsample was taken from the middle of the vessel using a syringe. The 

aqueous phases were extracted in a 1:1 (v:v) ratio with cylcohexane. The shaking time was 30 

seconds. The organic layer was analysed.

The pH of each of the water mixtures (except for the blank) was determined. The samples were diluted 

in a 9:1 (v:v) ratio with 0.1 M sodium chloride in order to obtain a high electrolyte concentration for 

accurate pH measurement.

10.4. Analytical method

10.4.1. Analytical conditions

Quantitative analysis was performed according to the analytical method as validated in Chapter 7.

Turbidity measurements:

Instrument 2100 AN IS Laboratory Turbidimeter (Hach Company, 

Loveland, Colorado, USA)

Unit NTU

Calibration range < 0.1 to 7500 NTU

10.4.2. Preparation of the calibration solutions

Stock solutions 

Stock solutions of the test substance were prepared in cyclohexane at concentrations of 2000 -

5000 mg/l. 

Calibration solutions

Calibration solutions in the concentration range of 0.2 – 20 mg/l were prepared from two stock 

solutions. The end solution of the calibration solutions was cyclohexane. 

10.4.3. Sample injections

Calibration solutions were injected in duplicate. Test samples were analysed by single injection. 

10.4.4. Calibration curves

Calibration curves were constructed using five concentrations. For each concentration, two responses 

were used. Linear regression analysis was performed using the least squares method with a 

1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.
FRET 09-0536 Project 504371

Final report - Page 26 -

10.5. Interpretation

10.5.1. Specifications

The water solubility of a test substance is specified by the saturation mass concentration of the test 

substance in water at a given temperature and is expressed in g/l.

10.5.2. Formulas

Response (R) Peak area of the test substance [units]

Maximum difference 100%

mean value

highest value lowest value × −

where:

'mean value' is the mean of the highest and lowest value.

Calibration curve R = a CN + b

where:

CN = nominal concentration [mg/l]

a = slope [units × l/mg]

b = intercept [units]

Analysed concentration (CA) d [mg/l]

a

(R b) CA × − =

where:

d = dilution factor

10.6. Results

10.6.1. Preliminary test 1

The content of test substance dissolved in the water sample after two and three centrifugation steps 

was 7.35 and 4.85 mg/l, respectively. The large difference of 41% between the analysed 

concentrations between these centrifugation steps indicates that the centrifugation method was not 

adequate for removal of undissolved test substance.

10.6.2. Preliminary test 2

Since the water solubility during preliminary test 1 was below the expected solubility according to 

information of the sponsor, the preliminary test was repeated with centrifugation was performed in 

polypropylene tubes as well as glass tubes. The content of test substance dissolved in the water 

sample after two, three and four centrifugation steps in polypropylene tubes was 7.37, 6.66 and 5.21

mg/l, respectively. The content of test substance dissolved in the water sample after two and three 

centrifugation steps in glass tubes was 10.6 and 10.1 mg/l, respectively.

10.6.3. Main study 1 – column elution method

Since the test substance concentrations observed during the preliminary tests was ≤ 10-2 g/l, the main 

study was started with the column elution method. The pH values of the water fractions that were 

collected for the determination of the flow rates were 8.1 at both flow rates. The turbidity of the eluates 

was < 0.2 NTU. According to this, no undissolved particles were detected in the eluates. No test 

substance was detected at either flow rate. Analysis of the column material indicated that the column 

contained test substance at very low loading at the start of the study, probably caused by evaporation 

of the test substance. 
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Final report - Page 27 - 

10.6.4. Main study 2 – column elution method

Because of the low column loading during the first column elution study, a second column elution 

study was performed with test substance dry mixed with the carrier material. The eluate from the test 

substance column was turbid. Analysis of this solution confirmed that a large amount of test substance 

elutes from the column. This indicated that the column elution method was not applicable for the 

determination of the water solubility of the test substance. Therefore, an additional water solubility 

study was performed based on the slow-stirring flask method.

10.6.5. Main study 3 –slow-stirring flask method

The results for the samples taken at 24, 48 and 72 hours are given in Table 6.

The 48-hour measurement was significantly lower than the 24- and 72- hour measurements . Based 

on this, the water solubility of the test substance was given as the mean value of the 24- and 72-hour 

measurements. The maximum difference between the 24- and 72-hour measurements was only 

slightly higher than the criterion of 15% (i.e. 18%) limiting the effect on the study outcome. 

Prolongation of the stirring time was not desirable considering reactivity of the test substance with 

oxygen as indicated by the sponsor.

No test substance was detected in the pretreated sample from the blank water mixture.

Table 6 Main study - water solubility of the test substance 

Stirring time

[hours]

Analysed 

concentration

[g/l]

Mean

[g/l]

MD

[%]

pH

24 0.0147 0.0134 18 7.3

48 0.008211 7.3

72 0.0122 7.3

1 Outlier, result was excluded.

10.7. Conclusion

The slow-stirring flask method was applied for the determination of the water solubility of 

FRET 09-0536. 

The water solubility of the test substance at 20°C was 0.0134 g/l. 

The pH of the aqueous samples was 7.3. 
FRET 09-0536 Project 504371

Final report - Page 28 - 

11. PARTITION COEFFICIENT

11.1. Guidelines

European Community (EC), EC no. 440/2008, Part A: Methods for the Determination of PhysicoChemical Properties, Guideline A.8: “Partition Coefficient”, Official Journal of the European Union no. 

L142, May 31, 2008.

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

of Chemicals no. 117: "Partition Coefficient (n-octanol/water), High Performance Liquid 

Chromatography (HPLC) Method", April 13, 2004.

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

OPPTS 830.7570: "Partition Coefficient (n-octanol/water), Estimation by Liquid Chromatography", 

August 1996.

11.2. Reagents

Water Tap water purified by a Milli-Q water purification system 

(Millipore, Bedford, MA, USA)

Methanol Biosolve, Valkenswaard, The Netherlands

All reagents were of analytical grade, unless specified otherwise.

11.3. Performance of the study

Four methods are available for the determination of the partition coefficient (Pow) of the test substance. 

The principle of each method is described below:

1. Shake flask method

Test substance is partitioned in a two-phase system of n-octanol and water. After phase separation, 

the concentration of the test substance in both phases is determined by a suitable analytical method. 

The shake flask method can be used for test substance with a log Pow value in the range of -2 to 4. 

2. HPLC method

A solution of the test substance is injected onto a reversed-phase HPLC column. The log Pow value is 

calculated from the retention time of the test substance. The HPLC method covers log Pow values in 

the range of 0 to 7.2. 

3. Estimation method

The solubility of the test substance in n-octanol and in water is determined. The quotient of the noctanol solubility and the water solubility is an estimation of the Pow. The estimation method can be 

applied if the shake flask method and HPLC method are not applicable.

4. Calculation method

The calculation method is based on the theoretical fragmentation of the molecule into suitable 

substructures for which reliable log Pow increments are known. The log Pow is obtained by summing the 

fragment values and the correction terms for intramolecular interactions. Lists of fragment constants 

and correction terms are available. 

The calculation method was used for a preliminary estimation of the Pow of the main component of the

test substance (see Appendix 2). Calculation was performed using the Rekker calculation method 

(PrologP 6.0, module in Pallas 3.0, CompuDrug International, San Francisco, CA, USA). Based on the 

calculation it was decided to perform the HPLC method for an accurate determination of the Pow. 
FRET 09-0536 Project 504371

Final report - Page 29 -

According to the guidelines, the determination of the Pow of the test substance should be performed in 

its non-ionised form. Therefore, the pKa values of the test substance were calculated using the Perrin 

calculation method (pKalc 5.0, module in Pallas 3.0, CompuDrug International). Based on the 

calculations, it was decided to perform the HPLC method at neutral pH.

Solutions of reference substances and the test substance were analysed. The capacity factor (k') of 

each compound was calculated from its retention time. The log k’ values of the references substances 

were plotted against the known log Pow values. A linear regression program was used to calculate the 

calibration curve. Linear regression analysis was performed using the least squares method. The 

coefficient of correlation (r) was calculated. The log Pow value for the test substance was calculated by 

substituting its mean log k’ in the calibration curve. The value of log Pow obtained from duplicate 

measurements was within ± 0.1 log units.

11.4. Analytical method 

11.4.1. Analytical conditions

Instrument Acquity UPLC system (Waters, Milford, MA, USA)

Detector Acquity UPLC TUV detector (Waters)

Column Acquity UPLC HSS T3, 100 mm × 2.1 mm i.d., 

dp = 1.8 μm (Waters)

Column temperature 35°C ± 1°C

Mobile phase neutral pH 75/25 (v/v) methanol/water

Flow 0.4 ml/min

Injection volume 5 μl

UV detection 210 nm

11.4.2. Preparation of the solutions 

Solution of the unretained compound

A 5.30 g/l stock solution of formamide (99.2%, [75-12-7], Alfa Aesar GmbH & Co KG, Karlsruhe, 

Germany) in methanol was used. The stock solution was diluted to obtain an end solution of 75/25

(v/v) methanol/water.

The formamide blank solution was 75/25 (v/v) methanol/water.

Reference substance solution with log Pow ≤ 6.5

Stock solutions of the reference substances at concentrations of approximately 1 g/l in methanol were 

used. The stock solutions were diluted to obtain an end solution of 75/25 (v/v) methanol/water. 

The blank solution for the mixture of reference substances was 75/25 (v/v) methanol/water. 

Reference substance Purity CAS number Supplier log Pow

1

Bromobenzene 99.8% 108-86-1 Acros Organics 3.0

1,4-Dichlorobenzene 99.9% 106-46-7 Acros Organics 3.4

Biphenyl 99.8% 92-52-4 Acros Organics 4.0

Dibenzyl 99.7% 103-29-7 Acros Organics 4.8

Triphenylamine 99.9% 603-34-9 Acros Organics 5.7

4,4’-DDT 98.2% 50-29-3 Supelco 6.5

1 values according to the OECD 117 guideline

Acros Organics, Geel, Belgium

Supelco, Bellefonte, PA, USA
FRET 09-0536 Project 504371

Final report - Page 30 - 

Test solution

A 1000 mg/l stock solution of the test substance was prepared in methanol. The stock solution was 

diluted in a 3:1 (v/v) ratio with water to obtain an end solution of 75/25 (v/v) methanol/water. The final 

concentration of the test substance solution was 750 mg/l.

The test substance blank solution was 75/25 (v/v) methanol/water.

11.4.3. Injections

The reference substance- and test substance solutions were injected in duplicate. Blank solutions 

were analysed by single injection. 

11.5. Interpretation

Capacity factor (k’)

0

0r

t

(t )t

k'

−

=

where:

tr = retention time

t0 = mean column dead time

Calibration curve = ow + bP log ak' log

where:

a = slope

b = intercept

11.6. Results

11.6.1. Calculation method

Calculation of the Pow

The Pow of the test substance was calculated to be 1.4 × 104 (log Pow 4.2) using the Rekker calculation 

method.

Calculation of the pKa

The following pKa values in the logarithm range of 1 - 14 for acidic and basic groups in the molecular 

structure of the test substance were calculated using the Perrin calculation method:

Acidic

RCHO pKa 13.82

11.6.2. HPLC Method 

UPLC chromatograms of the test substance solution and corresponding blank are shown in Figure 8.

In the chromatogram of the test solution the largest test substance response (95%) was caused by 12 

overlapping peaks between retention times of 2.40 and 4.20 minutes. Furthermore, eight minor 

impurities with retention times of 8 minutes and longer were observed. 

The results of the HPLC method are given in the Table 7. Figure 9 shows the calibration curve of the 

log k’ of the reference substances as function of log Pow. The equation of the regression line was: log 

k’ = 0.309 × log Pow – 0.703 (r = 0.998, n = 12).
FRET 09-0536 Project 504371

Final report - Page 31 -

Table 7 Partition coefficient of the test substance 

Substance tr,1

[min]

tr,2

[min]

mean tr

(n=2)

log Pow Pow Area

%

Formamide (t0) 0.583 0.581 0.582

Bromobenzene 1.516 1.515 3.0

1,4-Dichlorobenzene 1.886 1.885 3.4

Biphenyl 2.539 2.537 4.0

Dibenzyl 4.237 4.234 4.8

Triphenylamine 7.834 7.829 5.7

4,4’-DDT 11.279 11.274 6.5

TS205292 Peak 1 2.482 2.486 2.484 3.9 8.8 × 103

95 1

TS205292 Peak 2 2.666 2.659 2.663 4.1 1.2 × 104

TS205292 Peak 3 2.755 2.755 2.755 4.1 1.4 × 104

TS205292 Peak 4 2.862 2.861 2.862 4.2 1.6 × 104

TS205292 Peak 5 3.000 2.999 3.000 4.3 1.9 × 104

TS205292 Peak 6 3.111 3.110 3.111 4.3 2.2 × 104

TS205292 Peak 7 3.262 3.268 3.265 4.4 2.7 × 104

TS205292 Peak 8 3.438 3.437 3.438 4.5 3.3 × 104

TS205292 Peak 9 3.620 3.614 3.617 4.6 4.0 × 104

TS205292 Peak 10 3.836 3.833 3.835 4.7 5.0 × 104

TS205292 Peak 11 3.960 3.953 3.957 4.8 5.6 × 104

TS205292 Peak 12 4.078 4.082 4.080 4.8 6.3 × 104

TS205292 Peak 13 8.000 7.996 7.998 5.9 7.2 × 105 1.3

TS205292 Peak 14 8.255 8.217 8.236 5.9 8.0 × 105 0.60

TS205292 Peak 15 8.889 8.902 8.896 6.0 1.0 × 106 0.17

TS205292 Peak 16 9.813 9.840 9.827 6.2 1.5 × 106 0.51

TS205292 Peak 17 10.120 10.113 10.117 6.2 1.6 × 106 0.43

TS205292 Peak 18 12.625 12.616 12.621 6.5 2 3.5 × 106 0.50

TS205292 Peak 19 13.170 13.178 13.174 6.6 2 4.0 × 106 0.59

TS205292 Peak 20 13.825 13.825 13.825 6.72 4.7 × 106 0.62

1 Combined area % of Peak 1 – Peak 12

2 Extrapolated

11.7. Conclusion

The HPLC method was applied for the determination of the partition coefficient (Pow) of FRET 09-0536.

The Pow and log Pow value of the test substance and impurities at neutral pH was:

Pow log Pow Area

%

Test substance – peak 1-12 8.8 × 103 – 6.3 × 104 3.9 - 4.8 95

Test substance – peak 13 7.2 × 105 5.9 1.3

Test substance – peak 14 8.0 × 105 5.9 0.60

Test substance – peak 15 1.0 × 106 6.0 0.17

Test substance – peak 16 1.5 × 106 6.2 0.51

Test substance – peak 17 1.6 × 106 6.2 0.43

Test substance – peak 18 3.5 × 106 6.5 0.50

Test substance – peak 19 4.0 × 106 6.6 0.59

Test substance – peak 20 4.7 × 106 6.7 0.62
FRET 09-0536 Project 504371

Final report - Page 32 -

11.8. Figures

Figure 8 HPLC chromatograms of the 750 mg/l test solution [top; res. id. 1286] and 

corresponding blank [bottom; res. id. 1285].

Figure 9 The regression line of the reference substances: log k’ versus log Pow. TS205292 Peak 1 - 2.482 TS205292 Peak 2 - 2.666 TS205292 Peak 3 - 2.755 TS205292 Peak 4 - 2.862 TS205292 Peak 5 - 3.000 TS205292 Peak 6 - 3.111 TS205292 Peak 7 - 3.214 TS205292 Peak 8 - 3.383 TS205292 Peak 9 - 3.620 TS205292 Peak 10 - 3.836 TS205292 Peak 11 - 3.960 TS205292 Peak 12 - 4.039 TS205292 Peak 13 - 8.000 TS205292 Peak 14 - 8.255 TS205292 Peak 15 - 8.889 TS205292 Peak 16 - 9.813 TS205292 Peak 17 - 10.120 TS205292 Peak 18 - 12.625

TS205292 Peak 19 - 13.170

TS205292 Peak 20 - 13.825

AU 0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

AU 0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

Minutes

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

-0.8

-0.3

0.2

0.7

1.2

1.7

2.5 3 3.5 4 4.5 5 5.5 6 6.5 7

log k'

log Pow
FRET 09-0536 Project 504371

Final report - Page 33 -

Appendix 1 Certificate of analysis
FRET 09-0536 Project 504371

Final report - Page 34 - 

Appendix 2 Substance Identity Information Document for FRET 09-0536 (multi-constituent)

Identification (Trade 

Name)

FRET 09-0536 (multi-constituent)

Chemical (IUPAC) Name 

of multi-constituent:

Reaction mass of 3-[(2R,4s,6S)-tricyclo[5.2.1.02,6]dec-4-yl]propanal and 3-

[(2R,4r,6S)-tricyclo[5.2.1.02,6]dec-4-yl]propanal and 3-[(2R,6S)-

tricyclo[5.2.1.02,6]dec-3-yl]propanal

CAS no multiconstituent

Batch/Lot number

IPC number

Constituent 1 Constituent 2 Constituent 31

2-D Structure

IUPAC names of 

constituents

3-[(2R,6S)-

tricyclo[5.2.1.02,6]dec-3-

yl]propanal

3-[(2R,4s,6S)-

tricyclo[5.2.1.02,6]dec4-yl]propanal

3-[(2R,4r,6S)-

tricyclo[5.2.1.02,6]dec-4-

yl]propanal

CAS no

Concentration of tested 

sample

62% 11% 9%

Concentration range 57-70% 9-15% 6-15%

Molecular formula C13H20O C13H20O C13H20O

Molecular weight 192 192 192

 

1 Remove if only 2 constituents >10% are present; add additional Constituents if more than 3 are 

present.