# SCASSumika Chemical

. Analysis Service 

## Report
Study Title 1-7-5, Kikumoto-cho Niihama-shi, Ehime 792-0801 JAPAN Tel: +81-897-32-3411 Fax: +81-897-32-9644

Determination ofphysico-chemical properties of ·Hydrolysis as a Function ofpH ·Partition Coefficient (n-octanollwater) Study Director Shuichiro Manabe Study Completion Date May 7, 2013
Test Facility Sumika ChemicalAnalysis Service, Ltd. 1-7-5 Kikumoto-cho, Niihama-shi, Ehime, 792-0801 Japan StudyNumber

8316490-00

Sponsor

Sumika Chemical Analysis Service, Ltd.Study Number:8316490-00

Signatures

Operator Tsuyoshi Hamada

Date:

StudyDirector Shuichiro Manabe

Date:



### 1120Study Number:8316490-00
Contents 1. Purpose ofthe study...................................................................................................................... 3 2. Sample information....................................................................................................................... 3 3. Summary ........................................................................................................................................ 4 4. Hydrolysis as a Function ofpH..................................................................................................... 5 4.1. Guideline ................................................................................................................................. 5 4.2. Principle ofthe test ................................................................................................................ 5 4.3. Performance ofthe preliminary test..................................................................................... 5 4.3.1. Preparation ofapparatus and reagents......................................................................... 5 4.3.1.1 Preparation ofapparatus.......................................................................................... 5 4.3.1.2 Preparation ofbuffer solutions................................................................................. 5 4.3.1.3 Preparation ofmobile phase..................................................................................... 6 4.3.1.4 Preparation ofstandard solutions ........................................................................... 6 4.3.2. Pretreatment ................................................................................................................... 6 4.3.3. Analysis method .............................................................................................................. 6 4.4. Data handling......................................................................................................................... 6 4.5. Results..................................................................................................................................... 7 5. Partition coefficient (n-octanol/water).......................................................................................... 8 5.1. Guideline ................................................................................................................................. 8 5.2. Principle ofthe test ................................................................................................................ 8 5.3. Preparation of solutions ......................................................................................................... 8 5.3.1. Sample solutions.............................................................................................................. 8 5.3.2. Standard solutions .......................................................................................................... 8 5.3.3. Dead Time solution ......................................................................................................... 8 5.4. Analysis method ..................................................................................................................... 9 5.5. Data handling ......................................................................................................................... 9 5.6. Results ................................................................................................................................... 10 6. Tables............................................................................................................................................ 11 6.1. Hydrolysis as a Function ofpH ........................................................................................... 11 6.2. Partition coefficient (n-octanollwater)................................................................................. 12 7. Figures.......................................................................................................................................... 13 7.1. Hydrolysis as a function ofpH............................................................................................. 13 7.2. Partition coefficient (n-octanol/water)................................................................................. 17



### 2/20Study Number:8316490-00 1. Purpose ofthe study The purpose of the study was to determine the following physico-chemical properties for

·Hydrolysis as a Function ofpH ·Partition coefficient (n-octanollwater)
2. Sample information

Chemical Name: Structure Formula:

Identification: CAS No.: Purity: Lot No.: Appearance: Storage:



### 3/20Study Number:8316490-00 3. Summary Property Guideline Result Method Hydrolysis rate (50 

### "C, 5 days) Hydrolysis as a OECD 111
- Function ofpH < 10% (pH=4) < 10% (pH=7) < 10% (pH=9) Partition coefficient OECD 117 > 5.7 HPLCmethod (n-octanol/water)



### 4/204. Hydrolysis as a Function ofpH 4.1. Guideline The study was based on the following guideline: Study Number:8316490-00

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing ofChemicals no. 111: "Hydrolysis as a Function ofpH", April13, 2004. 4.2. Principle ofthe test Sterile aqueous buffer solutions of different pH values (pH 4, 7 and 9) are treated with the sample and incubated in the dark under controlled laboratory conditions (at constant temperatures).

After appropriate time intervals, buffer solutions are analyzed for the sample and for hydrolysis products. 4.3. Performance ofthe preliminary test 4.3.1. Preparation ofapparatus andreagents 4.3.1.1 Preparation of apparatus The glassware to be used was heated by drying machine. (170 - 180 

### 'C, 1 hour) The volumetric flasks which was after washed with 70 % ethanol and naturally dried were used. 4.3.1.2 Preparation ofbuffer solutions

(1) pH 4 buffer solution (0.1mol/ L phthalic acid buffer solution)

400 mL of water that was passing nitrogen for 5 min, 10.2 g of potassium hydrogen phthalate and 4 

### mL of0.1mo1/ 

### L sodium hydroxide were taken to the flask. Then water was added to prepare to 500mL, and stirred. (2) pH 7 buffer solution (0.1mol/ 

### L phosphatebuffer solution) 400 mL of water that was passing nitrogen for 5 min, 6.8 g of potassium dihydrogen phosphate and 29.6 mL of 1mol I L sodium hydroxide were taken to the flask. Then water was added to prepare to 500mL, and stirred. (3) pH 9 buffer solution (0.1mol/L borate buffer solution)

400 mL ofwater that was passing nitrogen for 5 min, 3.1 g ofboric acid and 21.3 mL of 1mo1/L sodium hydroxide were taken to the flask. Then water was added to prepare to 500mL, and stirred. Buffer solutions above were heatedin an autoclave. ( 121 

### 'C, 20 minutes) 

### 5/20Study Number:8316490-00 Preparation ofmobile phase The mobile phase was prepared to pH 2.1 by adding 0.1mol 

### I L phosphoric acid solution to a solution of sodium dihydrogen 0.1mol I L phosphate. 4.3.1.4 Preparation ofstandard solutions The standard solutions were prepared by diluting with the mobile phase within the range of0.14 to 3.6 giL. 4.3.2. Pretreatment

The sample were weighed and placed into each of 6 volumetric flasks (50 mL) and appropriate amount of 1mo1 

### I L sodium hydroxide were added to eachflasks. Then they were filled up to the marked line with buffer solutions. Two flasks each (pH 4, 7 and 9) were prepared at a concentration of 30 giL (approximately 0.01 mol 

### IL) withbuffer solutions.

The solutions were stood for 5 days in a water bath (50 

### ± 0.5 

### "C) and filtered through a 0.2-micrometer membrane filter, then the filtrates were diluted 20 times with mobile phase. The solutions were used as measurement solution.

4.3.3. Analysis method The solutions were analyzed with the GPC method. GPC conditions are shown below.

<GPC conditions>

Apparatus: Separation columns: LC-10ATVP (Shimadzu) Eluent: Flow rate: Detector: Column temperature: Injection volume: 4.4. Data handling Sample concentration of the exposed solution, hydrolysis rates of the sample and spike recoveries ofthe sample were calculated by the following equations:


### 6/20Y=aX+b C=f· X G = (C-D)iD • 100 H = (E-D)/D · 100
Y = Peak height (mV) X= Concentration ofmeasurement solution (giL) a= Slope b = y-Axis intercept f =Dilution factor C = Sample concentration ofthe exposed solution for 0 days. : Calculated from the measured values (giL) D = Sample concentration ofthe exposed solution Study Number:8316490-00

: Calculated from the amount ofthe adjusted the sample (giL) E =Sample concentration ofthe exposed solution for 5 days. : Calculated from the measured values (giL) G = Spike recoveries (%) H =Hydrolysis rate (%) 4.5. Results The results of standard solution measurement, spike recoveries and hydrolysis rate are shown inTable 1, 2 and 3.

The calibration curve is shown in Figure 1. The typical GPC chromatograms are shown in Figure 2 to 12 respectively. In each pH condition, spike recoveries were the average value of98% to 102% and variations were 0.26% to 6.8%.

In conclusion, the hydrolysis rate ofthe sample was determined tobe <10 %. Therefore, there is notnecessary ofthe further hydrolysis examination.



### 7/205. Partition coefficient (n-octanolfwater) 5.1. Guideline

The studywas based on the following guideline: Study Number:8316490-00

Organizationfor Economic Co-operation and Development (OECD), OECD Guidelines for the Testing ofChemicals no. 117: "Partition Coefficient (n-octanolfwater)", Aprill3, 2004. 5.2. Principle ofthe test Reverse phase HPLC IS performed on analytical columns packed with a commercially available solid phase containinglong hydrocarbon chains chemicallybound onto silica.

Hydrophilic chemicals are eluted first and lipophilic chemicals are eluted last. Elution corresponds to a partition coefficient (n-octanolfwater).

Partition coefficient (n-octanolfwater) of the sample is calculated with the retention time of standard materials. 5.3. Preparation ofsolutions

5.3.1. Sample solutions

The sample was dissolved in mixture ofborate buffer (pH9.8) I methanol (25/75) to obtain a sample solution at a concentration of**500~** 1000 mg/L. Thenthe solution was filtered through a 0.45-micrometer membrane filter.

As a blank solution, mobile phase was used. 5.3.2. Standard solutions

Aniline, toluene, naphthalene, ethyl benzene, biphenyl and triphenylamine were chosen as reference items from the list given in the OECD test guideline no.l17. They were dissolved into methanolto prepare a standard stock solution at a concentration of520 to 760 mg/L. The standard stock solutions were mixed and diluted with borate buffer (pH9.8) I methanol (25/75) to obtain a standard solution at a concentration between 26 and 38 mgiL respectively. 5.3.3. Dead Time solution

The stock solution of thiourea was prepared at a concentration of 590 mgiL with borate buffer (pH9.8) 

### I methanol (25/75). The dead time solution was prepared by diluting the stock solution to a concentration of29 mg/L withboratebuffer (pH9.8) I methanol (25/75).

8/205.4. Analysis method Apparatus: Study Number:8316490-00
LC-lOATVP (Shimadzu) Column: Mobile Phase: UV Detector: Injection Volume: Flow Rate: Column Temperature: 5.5. Data handling

The capacity factor is described bythe following equation. k = (tR-to)lto wherek = Capacity factor tR =Retention time ofthe sample and the standardmaterials to = Retention time ofthe dead time material The partition coefficient (n-octanollwater) of the sample is calculated by experimentally determiningits capacity factor and then inputtingk into the following equation.

Y=a·X+b whereY =Partition coefficient (n-octanol/water) (logP) X= logk a= Slope b = y-Axis intercept



### 9/20Study Number:8316490-00 5.6. Results

The calibration data ofpartition coefficient (n-octanol/water) are shown in Table 4. The calibration curve is shownin Figure 13. The coefficient ofdetermination (r2) was 0.963. In the case with no column, the increase of peak area of the sample solution was detected compared withtheblank solution. Howeverinthecase with a column, there was no significant difference in peak area between the sample solution and the blank solution. It presumed that the main components of was adsorbed in the reversed phase HPLC column and not eluted. Therefore, logP ofthe sample was presumed 5.7 or more.

The typical chromatograms of the dead time solution, the standard solution, the sample solution (with no column and with a column) and the blank solution (with no column and with a column) are shown in Figures 14, 15, 16 and 17 respectively.

In conclusion, the partition coefficient (n-octanol/water), logP, of the sample was presumed 5.7 or more.



### 10/20Study Number:8316490-00 6. Tables

6.1. Hydrolysis as a Function ofpH Table 1 The results ofstandard solution measurement

No. Concentration (giL) Height (mV)

1 0.14 13 2 0.56 52 3 1.4 132 4 3.6 325 Table 2 Boike recoveries Sample Sample concentration Preparation concentration Spike Difference Liquid Dilution Height in the Average Variation No. concentration in the liquid recoveries between exposure ratio (mV) measured (%) (%) (g/L) exposure (%) n1 and n2 solution (g/L)

(g/L) 0 day n=l 30 21 135 1.5 30 101 0.02 101 0.02 pH 4 n=2 30 21 135 1.5 31 101 0 day n=l 30 21 132 1.4 30 100 4.1 98 4.1 pH 7 n=2 30 21 126 1.4 29 96 0 day n=l 30 21 130 1.4 30 100 7.3 103 7.0 pH 9 n=2 30 21 139 1.5 32 107 Table 3 Hvdrolvsis rate Sample Sample Preparation concentration concentration Hydrolysis Liquid Dilution Height No. concentration in the measured in the liquid rate exposure ratio (mV) (g/L) solution exposure (%) (g/L) (g/L) 5 days n=1 30 20 133 1.5 30 <10 pH 4 n=2 30 21 139 1.5 31 (10 5 days n;::;l 30 21 130 1.4 29 (10 pH 7 n=2 30 21 136 1.5 31 <10 5 days n=1 30 21 143 1.6 33 <10 pH 9 n=2 30 21 139 1.5 32 <10 ll/20Study Number:8316490-00 6.2. Partition coefficient (n-octanol/water) Table 4 Calibration data ofuartition coefficient (n octanol/water) 

### - Dead time logk logPow Standard material to tR material <X> <Y> Thiourea 3.28

## ~ ..___________

### ~ Aniline 

# -----**~** 4.07 -0.616 0.9 Toluene 

# -----**~** 8.81 0.228 2.7 Naphthalene 

# -----**~** 10.68 0.354 3.6 Ethylbenzene

## ~**~** 11.30 0.389 3.2 Biphenyl 

# ----**~** 14.64 0.540 4.0 Triphenylamine 

# ----**~** 37.76 1.022 5.7



### 12/20Study Number:8316490-00 7. Figures

7.1. Hydrolysis as a function ofpH



### [mVl 350 300 250 

# fa> 200**~** _§_ 150 100

50 

# .,.

00.00 y ·0:1:'7tXT1-:-730S R'-noooo

## ~ 

# ./" ./" _/ 

# ../"" 1.00 2.00 Concentration (g/L) Figure 1 Calibration curve 3.00 

# _,.

4.00



### 300 00


### . 20[00 20. 00

400. 000 30. 00 [min] Figure 2 GPC chromatogram ofthe standard solution (No.3) 

### ' ' ' ' 

### 40. 00

300. 000**-----------------------------------~------------------------------------** ' ' ' ' ' ' 

### 200. 000**-----------------------------------~-------------------------------------** 

### 20. 00 ' 

### 30. 00 lminl Figure 3 GPC chromatogram ofthe mobile phase 

### 13/20 

### 40. 00Study Number:8316490-00 [mV]

[mVl

[mV] 

### 40!000 -----------------------------------,------------------------------------ ' ' ' ' ' 

### 300.000 ----------------------------------- '------------------------------------ 20!000 ----------------------------------- ------------------------------------ 20.00

400. 000 30. 00 [min] Figure 4 GPC chromatogram ofthe buffer solution (pH 4)

' 

# \I 40. 00

300. 000 -----------------------------------,------------------------------------ ' 

### 200. 000**-----------------------------------~** 

### 20. 00

400. 000 ' 

### y 30. 00 [mini Figure 5 GPC chromatogram ofthe buffer solution (pH 7) 

# \I 40. 00

300.000 ----------------------------------- ------------------------------------ 200. 000**-----------------------------------~------------------------------------** 

### 20. 00 ' ' 

### 30. 00 [mini Figure 6 GPC chromatogram ofthe buffer solution (pH 9)

14/20 

# \( 40. 00Study Number:8316490-00 

### [mVl

[mVl

[mV] 400. 300. 000 20[000 1 00·**~8.** 00 lminl Figure 7 GPC chromatogram ofthe sample solution (0 day, pH 4 n=ll

300.000 20[000 1 00. 

### 008 2.00 3[00 40. 00 [min] Figure 8 GPC chromatogram ofthe sample solution (0 day, pH 7 n=l)

300.000 200. 000 100.000 20. 00 30. 00 [min] Figure 9 GPC chromatogram ofthe sample solution (0 day, pH 9 n-1)



### 15/20 40. 00Study Number:8316490-00 

### [mVl

[mVl

[mVl 

### 300.0 201000 1 

## oo.**o~8.l-,oo-o**-------------::30:-:-:.**o~o--------'---'** 

### 40. 00 [min] Figure 10 GPC chromatogram ofthe sample solution (5 day, pH 4 n=ll



### 300. 000 200. 000 100. 

# 008 2 .00 30. 00 40. 00 [min] Figure 11 GPC chromatogram ofthe sample solution (5 day. pH 7 n=ll



### 300. 000 200. 000 100.000 ' 

### 20. 00 30. 00 [min] Figure 12 GPC chromatogram ofthe sample solution (5 day, pH 9 n=ll

16/20 

### 40. 00Study Number:8316490-00 7.2. Partition coefficient (n-octanollwater)**~** 0 "- 

### "' .!l 

# •**1------~"l** -1--------jY= 2.893x + 2.425 _ R

2 = 0.963
-0.700 -0.200 0.300 logk Figure 13 Calibration curve



### 17/20 0.800Study Number:8316490-00


### [mVl

[mVl 

## 150.**OOOir---;::::=====-=-;----:---------:-----~** Thiourea

' 

## 100. 000 --------------------L------------------------L------------------------

0 ' ' ' ' ' ' ' ' ' 

## 50. 000 --------------------r------------------------r------------------------



## 0. OOO•t=:"::;:::::==;===::;=:;:==;t===;===::;=:;:==;==;===::;=:;:==;===;====j 

## 0. 00 20. 00 40. 00 60. 00 [min] Figure 14 HPLC chromatogram ofthe dead time solution


## 300.**000-.----------~------~,**-----------c

# ~: Toluene 

# I : : 

# T 1-

# r=-=;J:.:_Ei.:•

# th.:..:yl:..::._be:..::nz.:..:en:..::._e~ ,------------

# -!-----------------------



# ---------l**:7-~-----** -I

# Triphe~nylamine 1------------- - ---

' ' ' ' 

## 200. 000 100. 000

' ' ' ' 

## 0. 000**F==:'=~="';=~;===r'**=;===;====;=====r"--="='f===;====;=====;o==;c===j 

## 0. 00 20. 00 40. 00 60. 00 [min] Figure 15 HPLC chromatogram ofthe standard solution



### 18/20Study Number:8316490-00



### [mVl

[mV] 

## 40. 000

0.**00** 

## QOO tOO 400

40. 000 lminl 

# "·

0. 000 000 tOO 400 [min]
Figure 16 HPLC chromatograms ofthe sample solution andblank solution (with no column)



### 19/20Study Number:8316490-00


### [mV]

[mVl 

## 40. 000 20. 000

0. 0. 00 20. 00 40. 00 60. 00 [min]

Blank solution 

## 40. 000 ' 

### ------,-----------------------·----------------------- 

## 20. 000-

---------------------- -t------------ -----------I----------------------- ' ' '**0.0001~~~-~~~~~~-~~~~~--~-** 

## 0. 00 20. 00 40. 00 60. 00 [min[
Figure 17 HPLC chromatograms ofthe sample solution andblank solution (with a column)

20/20