REPORT

D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]:

Determination of Partition Coefficient (n-Octanol/Water)

Study Director: J A Walker

Test Facility: Envigo Research Limited

Shardlow Business Park

Shardlow

Derbyshire

DE72 2GD

UK

Sponsor: Univar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA

Study Monitor: Greenwich Chemical Consulting, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens

FL 33410

UNITED STATES OF AMERICA

Study Number: 41401172

Study Completion Date: 24 September 2015

On 21 September 2015 the legal entity Harlan Laboratories Ltd. was renamed as Envigo Research 

Limited. No other changes to the legal entity have occurred.

Page 1 of 23
Study Number: 41401172 Report Page 2

CONTENTS

CONTENTS....................................................................................................................................2

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE ....................................................3

QUALITY ASSURANCE STATEMENT......................................................................................4

SUMMARY ....................................................................................................................................5

GENERAL INFORMATION .........................................................................................................6

Schedule.................................................................................................................................6

Deviations from Study Plan...................................................................................................6

Archiving ...............................................................................................................................6

1 INTRODUCTION AND PURPOSE.....................................................................................7

1.1 Guidelines / Regulations..........................................................................................7

2 TEST ITEM ...........................................................................................................................8

3 TEST......................................................................................................................................9

3.1 Partition Coefficient (n-Octanol/Water)..................................................................9

3.1.1 Test System ...............................................................................................9

3.1.2 Performance of the Test.............................................................................9

3.1.3 Calculation...............................................................................................11

3.1.4 Results.....................................................................................................13

3.1.5 Discussion................................................................................................17

3.1.6 Conclusion...............................................................................................18

APPENDICES...............................................................................................................................19

Appendix 1 Certificate of Analysis...........................................................................................19

Appendix 2 Typical Chromatography.......................................................................................20

Appendix 3 Monitoring Authority Statement of GLP Compliance ..........................................23
Study Number: 41401172 Report Page 3

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE

Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Study Number:

Study Title:

41401172

D-erythro-Hex-2-enonic acid, .gamma-lactone,

monosodium salt:

Determination of Partition Coefficient (n-Octanoll

Water)

This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory

Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are

in accordance with GLP standards published as OECD Principles on Good Laboratory Practice

(revised 1997, ENV/MCICHEM(98)17); and are in accordance with, and implement, the

requirements of Directives 2004/9/EC and 200411O/EC.

These principles are compatible with Good Laboratory Practice regulations specified by

regulatory authorities throughout the European Community, the United States (EPA and FDA),

and Japan (MHLW, MAFF and METI).

This report fully and accurately reflects the procedures used and data generated. There were no

circumstances considered to have affected the integrity of the study or the validity of the data.

Study Director: J A Walker

Date:

2 4 SEP 2015
Study Number: 41401172 Report Page 4

QUALITY ASSURANCE STATEMENT

Study Number:

Study Title:

41401172

D-erythro-Hex-2-enonic acid, .gamma-lactone,

monosodium salt:

Determination of Partition Coefficient (n-Octanoll

Water)

The general facilities and activities are inspected at least once a year and the results are reported

to the relevant responsible person and management.

Study-related procedures conducted at the test facility were audited and inspected. The details of

these audits and inspections are given below.

Reported to the relevant

Dates and Types of QA Inspections Study Director and Test

Facility Management

Date of Inspection Type of Inspection Phase Inspected Report Date

18 June 2014 Study Plan Verification N/A 18 June 2014

14 January 2015 Process - based Partition Coefficient 14 January 2015

01 April 2015 Report Audit N/A 01 April 2015

This statement confirms that this final report reflects the raw data and the procedures followed.

Quality Assurance:

~ \-'\.~~"''''«_

.......\:\~

Date: 2 9 SEP 2015
Study Number: 41401172 Report Page 5

SUMMARY

The partition coefficient (n-octanol/water) of D-erythro-Hex-2-enonic acid, .gamma.-lactone, 

monosodium salt [CAS RN 6381-77-7] has been determined to be less than 2.84 x 10-2 at 

21.0 ± 1.0 °C, log10 Pow less than -1.55, using the shake-flask method, designed to be compatible 

with Method 107 of the OECD Guidelines for Testing of Chemicals, 27 July 1995.
Study Number: 41401172 Report Page 6

GENERAL INFORMATION

Schedule

Experimental Starting Date: 19 November 2014

Experimental Completion Date: 05 March 2015

Deviations from Study Plan

There were no deviations (unplanned changes) from the study plan.

Archiving

Unless instructed otherwise by the Sponsor, the study plan, all raw data (paper and electronic) 

and the final report will be retained in the Envigo Research Limited, Shardlow, UK archives for 

five years after which instructions will be sought as to further retention or disposal. Further 

retention or return of the data will be chargeable to the Sponsor.

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Study Number: 41401172 Report Page 7

1 INTRODUCTION AND PURPOSE

The purpose of this study is to evaluate the partition coefficient (n-octanol/water) of the test 

item.

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) 

Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087.

1.1 Guidelines / Regulations

The test methods described are designed to be compatible with the procedures indicated by the 

following internationally accepted guidelines and recommendations:

• OECD Guidelines for Testing of Chemicals (1995) No. 107 “Partition Coefficient

(n-octanol/water): Shake Flask Method”.
Study Number: 41401172 Report Page 8

2 TEST ITEM

Information as provided by the Sponsor. The Certificate of Analysis provided by the Sponsor is 

presented as Appendix 1.

Identification: D-erythro-Hex-2-enonic acid, .gamma.-lactone, 

monosodium salt

Appearance/Physical State: White granular solid

Lot: 14031314

Purity: 99.19 %

Expiry Date: 26 March 2016

Storage Conditions: Room temperature in the dark
Study Number: 41401172 Report Page 9

3 TEST

3.1 Partition Coefficient (n-Octanol/Water)

3.1.1 Test System

The determination was carried out using the shake-flask method, designed to be compatible with 

Method 107 of the OECD Guidelines for Testing of Chemicals, 27 July 1995.

The test system consisted of an aqueous and an n-octanol phase in the same vessel. A defined 

amount of test item was added (pre-dissolved in the appropriate phase). Partition of the test item 

between both phases was achieved by inversion of the mixture. The temperature was in the 

range of 20 °C to 25 °C and kept constant (± 1 °C).

Testing was performed under non-actinic lighting and amber glassware was used, to ensure that 

any photodegradation was kept to a minimum.

Purified (reverse osmosis) water was used during testing since it was determined though stability 

assessments that the test item is most stable in water with low hardness. In addition, the purified 

water contained nearly no dissolved oxygen which helped to minimize degradation of the test 

item.

The test item was found to have very limited stability in water. Through stability assessments, it 

was determined that a small amount of acetic acid sufficiently stabilizes the material in water. 

For this reason, acetic acid was used to prepare the organic phase and aqueous phase samples for 

analysis. The use of acetic acid did not have an adverse effect on the test system or test results. 

It is noted that during preliminary assessments where acetic acid was not used, no measurable 

recovery of the test item could be obtained.

3.1.2 Performance of the Test

Stock solutions were prepared by diluting test item [0.3804 g to 200 mL, 0.9501 g to 500 mL, 

and 0.3804 g to 200 mL; for the 1:1, 1:2 and 2:1 ratios, respectively) with n-octanol saturated 

water [containing 1 % by volume of a commercial pH 7 (phosphate) buffer solution].
Study Number: 41401172 Report Page 10

Six partitions (see following table) were performed. In each test, the combined volume of both 

phases occupied not less than 90% of the total volume of the test vessel.

Table 3.1

Test Organic/Aqueous 

volume ratio

Volume of water 

saturated n-octanol 

(mL)

Volume of stock 

solution (mL) Temperature (oC)

1

1:1

85 85 21.4

2 85 85 21.4

3

1:2

56 112 20.6

4 56 112 20.6

5

2:1

112 56 20.6

6 112 56 20.6

The shaking was performed by inversion of the flasks through approximately 180° over a five 

minute period. Phase separation was then obtained by centrifugation (for 15 minutes at 

13000 rpm at 20 °C). Aliquots of both phases were taken for analysis.

3.1.2.1 Analysis of the sample solutions

The concentration of test item in the sample solutions was determined by high performance 

liquid chromatography (HPLC).

Organic phase samples

An aliquot (10 mL) was extracted with a portion (10 mL) of 5 % acetic acid.

Aqueous phase samples

The samples were diluted by a factor of 20 using 5 % acetic acid.

Duplicate aliquots (A and B) of the stock solution were diluted by a factor of 20 using 5 % acetic 

acid.

Standards

A set of standard solutions of test item were prepared in 5 % acetic acid covering a nominal 

concentration range of 50 to 200 mg/L, with a duplicate at nominally 100 mg/L.

Matrix blank

5 % acetic acid
Study Number: 41401172 Report Page 11

Analysis

The standard and sample solutions were analyzed by HPLC using the following conditions:

HPLC System : Agilent Technologies 1100 Series

Detector type : Ultra-violet (UV)

Column : Thermo Scientific Acclaim Trinity P1 3μm 

(100 x 3.0 mm id)

Column temperature : 30 °C

Mobile phases : A: Acetonitrile

B: 200 mM ammonium acetate, adjusted to ~ pH 4 

with acetic acid

Gradient:

Time 

(mins)

%A % B

0 60 40

2 60 40

5 90 10

Flow-rate : 0.5 mL/min

Injection volume : 5 μL

UV detector wavelength : 267 nm

Retention time : ~ 2.1 mins

3.1.3 Calculation

The peak area and concentration of each standard were plotted on a calibration curve and the 

sample concentration interpolated from the curve. The concentration was then corrected for 

dilution factor.

From this curve, the concentration of test item in the injected solutions was calculated by the 

following equation:

Equation 3.1

Y = b . x + a ⇔ x = (y – a) / b

c = f . x

where:

y = peak area of injected sample solutions

x = concentration of injected sample solution [mg/mL]

b = slope

a = y-axis intercept

c = concentration of the test item [mg/L]

f = factor of dilution (1, for the organic phases; 20, for the aqueous phases)
Study Number: 41401172 Report Page 12

The total weights (mg) of test item in the respective phases were calculated using Equation 3.2, 

Equation 3.3 and Equation 3.4 respectively.

Equation 3.2

1000

V

W C x org

org = org

Equation 3.3

1000

V

W C x aq

aq = aq

Equation 3.4

1000

V W C x stock

stock = stock

The partition coefficient for each determination was calculated using Equation 3.5.

Equation 3.5

C

C

P

aq

org

ow =

where:

Cstock = analyzed stock solution concentration (mg/L)

Corg = analyzed organic phase concentration (mg/L)

Caq = analyzed aqueous phase concentration (mg/L)

Wstock = weight of test item found in the stock solution (mg)

Worg = weight of test item found in the organic phase (mg)

Waq = weight of test item found in the aqueous phase (mg)

Vstock = volume of stock solution used in the determination (mL)

Vorg = volume of organic phase used in the determination (mL)

Vaq = volume of aqueous phase used in the determination (mL)

Pow = partition coefficient
Study Number: 41401172 Report Page 13

3.1.4 Results

The mean peak areas obtained for the standard, stock and sample solutions are shown in the 

following two tables:

Table 3.2

Solution Mean Peak Area

Standard 50.3 mg/L 4.686 x 106

Standard 75.4 mg/L 1.052 x 107

Standard 101 mg/L 1.788 x 107

Standard 102 mg/L 1.941 x 107

Standard 151 mg/L 3.105 x 107

Matrix blank None detected

Organic Sample 1 < 2.30 x 106

Organic Sample 2 < 2.30 x 106

Aqueous Stock A 2.851 x 107

Aqueous Sample 1 1.075 x 107

Aqueous Sample 2 1.722 x 107

Aqueous Stock B 3.137 x 107

Standard 50.3 mg/L 7.028 x 106

Standard 75.4 mg/L 1.331 x 107

Standard 101 mg/L 2.085 x 107

Standard 101 mg/L 2.073 x 107

Standard 151 mg/L 3.587 x 107

Matrix blank None detected

Organic Sample 3 < 3.50 x 106

Organic Sample 4 < 3.50 x 106

Aqueous Stock A 1.932 x 107

Aqueous Sample 3 1.880 x 107

Aqueous Sample 4 2.008 x 107

Aqueous Stock B 2.002 x 107
Study Number: 41401172 Report Page 14

Table 3.3

Solution Mean Peak Area

Standard 50.7 mg/L 8.209 x 106

Standard 76.0 mg/L 1.473 x 107

Standard 101 mg/L 2.298 x 107

Standard 101 mg/L 2.155 x 107

Standard 152 mg/L 3.848 x 107

Matrix blank None detected

Organic Sample 5 < 4.10 x 106

Organic Sample 6 < 4.10 x 106

Aqueous Stock A 1.935 x 107

Aqueous Sample 5 1.829 x 107

Aqueous Sample 6 1.883 x 107

Aqueous Stock B 2.085 x 107

The calibration curves from which the sample concentrations were interpolated are shown in 

Figure 3.1, Figure 3.2 and Figure 3.3.

Figure 3.1 – Samples 1 and 2
Study Number: 41401172 Report Page 15

Figure 3.2 – Samples 3 and 4

Figure 3.3 – Samples 5 and 6
Study Number: 41401172 Report Page 16

The total weights (mg) and analyzed concentration (mg/L) of the respective phases are shown in 

the following table:

Table 3.4

Sample 

Number

Total 

Weight 

(mg)*

Organic Phase Aqueous Phase

% 

Recovery Analyzed 

Concentration (mg/L)

Weight 

(mg)†

Analyzed 

Concentration (mg/L)

Weight 

(mg)† pH

1 247 < 41.6 < 3.54 1.47 x 103 125 6.7 51.9

2 247 < 41.6 < 3.54 1.95 x 103 166 6.7 68.6

3 215 < 40.0 < 2.24 1.86 x 103 208 6.8 97.9

4 215 < 40.0 < 2.24 1.95 x 103 218 6.8 103

5 104 < 39.3 < 4.41 1.73 x 103 96.9 6.9 97.8

6 104 < 39.3 < 4.41 1.77 x 103 98.9 6.9 99.7

pH of stock solutions: 7.0 to 7.1 

Temperature: 21.0 ± 1.0 °C

The partition coefficient determined for each sample is shown in the following table:

Table 3.5

Sample Number Organic/Aqueous 

Volume Ratio Partition Coefficient Log10 Pow

Overall Partition 

Coefficient

1

1:1

< 2.84 x 10-2 < -1.55

< 2.84 x 10-2

2 < 2.13 x 10-2 < -1.67

3

1:2

< 2.15 x 10-2 < -1.67

< 2.15 x 10-2

4 < 2.05 x 10-2 < -1.69

5

2:1

< 2.27 x 10-2 < -1.64

< 2.27 x 10-2

6 < 2.23 x 10-2 < -1.65

Overall Pow : < 2.84 x 10-2 log10 Pow : < -1.55

* From analysis of the stock solution

† From analysis of the respective phase

 
Study Number: 41401172 Report Page 17

3.1.5 Discussion

As the test item is a salt, testing was performed using the shake-flask method at approximately 

pH 7, as indicated by test guidance. Based on the estimated pKa’s, the test item would be in its 

salted form.

Testing was performed under non-actinic lighting and amber glassware was used, to ensure that 

any photodegradation was kept to a minimum.

During method development, the test item was noted to degrade readily at room temperature and 

neutral pH; however, the test item appeared to be much more stable at low pH. Therefore, it was 

considered that performing the individual ratio samples on separate days and diluting / extracting 

with dilute acetic acid would a) quicken up the partitioning, and b) ensure that any degradation 

(hydrolysis / oxidation) of the test item was kept to a minimum.

The stock solution was prepared in n-octanol saturated water in place of water saturated

n-octanol. This was due to the insolubility of the test item in the organic phase. This deviation 

to the guideline has been considered to have negligible impact on the overall result and has been 

considered to be more appropriate to use this solvent to obtain a more realistic partition 

coefficient value.

The limit values for the organic samples were calculated based on the estimated limit of 

quantitation (LOQ) from the respective calibration curve. From the curves, it was considered 

that the LOQ was approximately half of the area obtained for the lowest calibration standard in 

each curve.

The recoveries observed for Samples 1 and 2 were noted to be low. Since these were the first 

two samples to be prepared, it is likely that these took somewhat more time to complete 

compared to the later samples. That said, the log10 Pow results for Samples 1 and 2 were similar 

to those obtained for Samples 3 to 6. Therefore, it was concluded that all six of the samples 

would be used in the calculation of the overall partition coefficient.

Based on the 98 to 103% recoveries determined for Samples 3 to 6, it was considered that the 

extraction (solvent-exchange) step for the organic phases was essentially 100%.

The relative stability of the test item in the organic phases compared to the stability in the 

aqueous phase was not evaluated. However, it was considered that the stability is most likely not 

solvent-dependent, and therefore, the rate of degradation in both phases would be similar.

Due to all six of the samples yielding a “less than” Pow value, the highest of all six of the sample 

results has been reported as the overall “less than” value.
Study Number: 41401172 Report Page 18

3.1.6 Conclusion

The partition coefficient of the test item has been determined to be less than 2.84 x 10-2 at 

21.0 ± 1.0 °C, log10 Pow less than -1.55.
Study Number: 41401172 Report Page 19

APPENDICES

Appendix 1 Certificate of Analysis
Study Number: 41401172 Report Page 20

Appendix 2 Typical Chromatography

Standard 152 mg/L

Minutes

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

mAU

0

100

200

300

2.115

VWD: Signal A, 267 nm

PC00948 006 Vial 6 Standard L2.dat

Retention Time

Standard 50.7 mg/L

Minutes

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

mAU

0

20

40

60

2.122

VWD: Signal A, 267 nm

PC00948 002 Vial 2 Standard L5.dat

Retention Time
Study Number: 41401172 Report Page 21

Organic Sample 5

Minutes

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

mAU

0

10

20

30

40

50 VWD: Signal A, 267 nm

PC00948 007 Vial 7 Org 5.dat

Retention Time

Aqueous Sample 5

Minutes

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

mAU

0

25

50

75

100

125

2.117

VWD: Signal A, 267 nm

PC00948 010 Vial 10 Aq 5.dat

Retention Time
Study Number: 41401172 Report Page 22

Matrix Blank

Minutes

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

mAU

0

10

20

30

40

50 VWD: Signal A, 267 nm

PC00948 001 Vial 1 Diluent B.dat

Retention Time
Study Number: 41401172 Report Page 23

Appendix 3 Monitoring Authority Statement of GLP Compliance 

On 21 September 2015 the legal entity Harlan Laboratories Ltd. was renamed as Envigo Research Limited. No 

other changes to the legal entity have occurred.