## 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 23Study Number: 41401172 R eport Page 2



## CONTENTS

CONTENTS

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

STUDY DIRECTOR STATE MENT OF GLP COMPLIAN CE

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

QUALITY ASSURANCE ST ATEMENT

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

SUMMAR Y

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

GENERAL INFORMATION

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

Schedule

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

Deviations from Study Plan

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

Archiv ing

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

1 INTRODUCTION AND PUR POSE

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

1.1 Guidelines / Regulations

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

2 TE ST 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

................................ .......... 23Study 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

(re v ised 1997, ENV

/ MC I CHEM(98)17); and are in accordance with, and implement, the

requirements of Directives 2004 / 9 / EC and 200411

O / EC

.

These principles are compatible with Good Laboratory Practice regulations specified by

regulatory authorities throughout the European Community, t he 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 2015Study 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

- Octanol l

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 

## 2015Study Number: 41401172 R eport 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, log 10 P

ow less than

- 1.55 , using the shake

- flask method, designed to be compatible

with Method 107 of the OECD Guidelines for Tes ting of Chemicals, 27 July 1995

.Study Number: 41401172 R eport 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, U K 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 writ ten consent

.Study Number: 41401172 R eport Page 7



## 1 INTRODUCTION AND PUR POSE

The purpose of this study is to evaluate the p artition coefficient (n

- octanol/water) of the test

item.

This study is being performed based on the U.S. Toxic Substa nces 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 R eport 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 darkStudy Number: 41401172 R eport Page 9



## 3 TEST

3.1 Partition Coefficient (n

- Octanol/Water)

3.1.1 Test S ystem

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 consist ed 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

a ny 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

S tock solution s 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 R eport Page 10

Six partitions (see following table) were p erformed. 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 p erformance

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

.

S tandards

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 acidStudy Number: 41401172 R eport 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 phase s : 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

w here:

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 ph ases)Study Number: 41401172 R eport 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

x C W org

org org =

Equation 3

. 3

1000 V

x C W aq

aq aq =

Equation 3

. 4

1000 V

x C W stock

stock stock =

The partition coefficient for each determination was calculated using Equation 3

. 5

.

Equation 3

. 5

C C
P

aq org

ow =

w here:

C stock = analyzed stock solution concentration (mg/L)

C org = analyzed organic phase concentration (mg/L)

C aq = analyzed aqueous phase concentration (mg/L)

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

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

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

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

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

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

P

ow = partition coeffic ientStudy Number: 41401172 R eport Page 13

3.1.4 Results

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

following two table s :

Table 3

. 2

Solution Mean Peak Area

Standard 50.3 mg/L 4.686 x 10 6

Standard 75.4 mg/L 1.052 x 10 7

Standard 101 mg/L 1.788 x 10 7

Standard 102 mg/L 1.941 x 10 7

Standard 151 mg/L 3.105 x 10 7

Matrix blank None detected

Organic Sample 1 < 2.30 x 10 6

Organic Sample 2 < 2.30 x 10 6

Aqueous Stock A 2.851 x 10 7

Aqueous Sample 1 1.075 x 10 7

Aqueous Sample 2 1.722 x 10 7

Aqueous Stock B 3.137 x 10 7

Standard 50.3 mg/L 7.028 x 10 6

Standard 75.4 mg/L 1.331 x 10 7

Standard 101 mg/L 2.085 x 10 7

Standard 101 mg/L 2.073 x 10 7

Standard 151 mg/L 3.587 x 10 7

Matrix blank None detected

Organic Sample 3 < 3.50 x 10 6

Organic Sample 4 < 3.50 x 10 6

Aqueous Stock A 1.932 x 10 7

Aqueous Sample 3 1.880 x 10 7

Aqueous Sample 4 2.008 x 10 7

Aqueous Stock B 2.002 x 10 7Study Number: 41401172 R eport Page 14

Table 3

. 3

Solution Mean Peak Area

Standard 50.7 mg/L 8.209 x 10 6

Standard 76.0 mg/L 1.473 x 10 7

Standard 101 mg/L 2.298 x 10 7

Standard 101 mg/L 2.155 x 10 7

Standard 152 mg/L 3.848 x 10 7

Matrix blank None detected

Organic Sample 5 < 4.10 x 10 6

Organic Sample 6 < 4.10 x 10 6

Aqueous Stock A 1.935 x 10 7

Aqueous Sample 5 1.829 x 10 7

Aqueous Sample 6 1.883 x 10 7

Aqueous Stock B 2.085 x 10 7

Th e calibration curve s 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 2Study Number: 41401172 R eport Page 15

Figure 3

. 2

– Samples 3 and 4

Figure 3

. 3

– Samples 5 and 6Study Number: 41401172 R eport Page 16

The total weights (mg) and analyze d 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 Analyze d

Concentration (mg/L) Weight

(mg) † Analyze d

Concentration (mg/L) Weight

(mg)† pH

1 247 < 41.6 < 3.54 1.47 x 10 3 125 6.7 51.9

2 247 < 41.6 < 3.54 1.95 x 10 3 166 6.7 68.6

3 215 < 40.0 < 2.24 1.86 x 10 3 208 6.8 97.9

4 215 < 40.0 < 2.24 1.95 x 10 3 218 6.8 103

5 104 < 39.3 < 4.41 1.73 x 10 3 96.9 6.9 97.8

6 104 < 39.3 < 4.41 1.77 x 10 3 98.9 6.9 99.7

pH of stock solution s : 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 Log 10 P

ow 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 P

ow : < 2.84 x 10 - 2 log 10 P

ow : <

- 1.55

* From analysis of the stock solution

† From analysis of the respective phaseStudy Number: 41401172 R eport 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 pK a ’s, the test item w ould be in its

salted form.

Testing was performed under non

- actinic lighting and amber glassware was used, to ensure that

any photodegra dation 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 ind ividual 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 prepa red in n

- octanol saturated wa ter 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 be en 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 sample s to be prepared, it is likely that these took somewhat more time to complete

compared to the later samples. That said, the log 10 P

ow 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 degradati on in both phases would be similar.

Due to all six of the samples yielding a “less than” P

ow value, the highest of all six of the sample

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

3.1.6 Conclusion

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

21.0 ± 1.0 °C, log 10 P

ow less than

- 1.55.Study Number: 41401172 R eport Page 19



## APPENDI CES

Appendix 1 Certificate of AnalysisStudy Number: 41401172 R eport 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 5.0mAU

0 100 200 3002.115 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 5.0mAU

0 20 40 602.122 VWD: Signal A, 267 nm

PC00948 002 Vial 2 Standard L5.dat

Retention TimeStudy Number: 41401172 R eport 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 5.0mAU

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 5.0mAU

0 25 50 75 100 1252.117 VWD: Signal A, 267 nm
PC00948 010 Vial 10 Aq 5.dat

Retention TimeStudy Number: 41401172 R eport 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 5.0mAU

0 10 20 30 40 50

VWD: Signal A, 267 nm

PC00948 001 Vial 1 Diluent B.dat

Retention TimeStudy Number: 41401172 R eport 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

.