THE DOW CHEMICAL COMPANY STUDY ID: PAGE 1 OF 15

STUDY TITLE

DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 GUIDELINE

Test Guidelines
OECD Guideline 111 (2004)

Author(s)

Study Completion Date 26 May 2011

Sponsor The Dow Chemical Company

Midland, Michigan 48674

Performing Laboratory Toxicology & Environmental Research and Consulting The Dow Chemical Company

Midland, Michigan 48674

Laboratory Project Study IDTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 5 TABLE OF CONTENTS

Page
STUDY TITLE ....................................................................................................................1

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ..................2
QUALITY ASSURANCE STATEMENT ..........................................................................3

SIGNATURE PAGE ...........................................................................................................4

SUMMARY .........................................................................................................................6

INTRODUCTION ...............................................................................................................7 Purpose ............................................................................................................................7 Test Guidelines ................................................................................................................7 Quality Assurance ...........................................................................................................7 Archiving ........................................................................................................................7 Safety

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

MATERIALS .......................................................................................................................7 Test Material ...................................................................................................................7 Chemical Name ...............................................................................................................7 Synonyms ........................................................................................................................7 Supplier, City, State (Lot, Reference Number) ...............................................................7 Purity/Characterization (Method of Analysis and Reference) ........................................8 Characteristics .................................................................................................................8

Molecular Formula .......................................................................................................8 Molecular Weight .........................................................................................................8 Chemical Structu re .......................................................................................................8 Previous Information .......................................................................................................8

EXPERIMENTAL DESIGN ...............................................................................................9 Analytical Procedures .....................................................................................................9

STUDY SPECIFIC PARAMETERS .................................................................................10 Data and Reporting .......................................................................................................10

STATISTICS .....................................................................................................................10

RESULTS AND DISCUSSION ........................................................................................10

CONCLUSIONS ................................................................................................................11

REFERENCES ..................................................................................................................12

TABLES ............................................................................................................................13 Table 1. Analytical Testi ng Conditions and Settings........................................................13

Table 2. Hydrolysis Results .............................................................14

FIGURE .............................................................................................................................16 Figure 1. Ln Concentration vs Time ...............................................16THE DOW CHEMICAL COMPANY STUDY ID: PAGE 6

SUMMARY
A 1st tier hydrolysis study was conducted for in accordance with the test procedure described in the OECD “Hydrolysis as a Function of pH” guideline (method 111). Precautions were taken to minimize possible biodegradation, photodegradation, and oxidation reactions. Buffered solutions containing 3 μg /L of at pH 4, 7, and 9 were incubated at 50 ± 0.5°C for five days in the dark. Solutions containing 3 μg /L of at pH 1.2 were incubated at 37
± 0.5°C for 17 hours in the dark. concentrations in the pH 4, 7 and 9 test solutions were determined on day 0, 1, and 5 by reverse phase high performance liquid chromatography with MS detection (HPLC

- MS). concentrations in the pH 1.2 test solutions were determined for 0, 1, 2 and 17 hours by
HPLC

- MS.
exhibited increasing hydrolysis rates with increasing pH. No detectable degradation of the test substance occurred after 17 hours at pH 1.2 at 37oC. After five days at 50°C, degradation of the test substance was less than 7 % at p H 4, 38 % at pH 7, and 100 % at pH 9. Corresponding half lives of the test substance were >1 year at pH 4 , 7.5 days at pH 7 , and <1 day at pH 9. Since less than 10% degradation occurred after 5 days at pH 4, the test substance is considered hydrolytically stable at pH 4

.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 7

INTRODUCTION

Purpose
The objective of this study was to investigate the hydrolysis of following the guidelines of the Organisation for Economic Co-Operation and Development (OECD) method 111 “Hydrolysis as a Function of pH” ( OECD, 2004 ).

Test Guidelines

OECD Organisation for Economic Co-operation and Development, (OECD) Guidelines for Testing of Chemicals . Guideline Number 111: Hydrolysis as a Function of pH. 13 April 2004.
Quality Assurance
The study conduct, data, protocol, protocol changes/revisions, and f inal report were inspected by the Quality Assurance Unit, Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.
Archiving
The data, protocol, protocol changes/revisions, and final report were archived by the
Toxic ology & Environmental Research and Consulting archivist and stored at The Dow Chemical Company, Midland, Michigan.
Safety
All personnel involved in the study were advised of the safety precautions to follow when handling the test material. Chemical safety information was made available.

MATERIALS

Test Material

Chemical Name

Synonyms

Supplier, City, State (Lot, Refer ence Number)
The Dow Chemical Company, Midland, Michigan (Lot# BatchTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 8

Purity/Characterization (Method of Analysis and Reference)
The purity of the test material was determined to be by gas chromatography with identification by Fourier transform infrared spectroscopy and gas chromatography mass spectrometry

Characteristics

Molecular Formula

Molecular Weight

Previ ous Information
Hydrolysis can be an important abiotic transformation process for chemicals that enter the aqueous environment. A tiered testing approach is recommended by the OECD to assess the hydrolytic fate of a compound in water. A preliminary 1 st tier study is usually conducted at 50°C to determine the susceptibility of the test compound to hydrolysis at pH 4, 7, and 9. Examination of hydrolysis at pH 1.2 may also be included if a physiologically relevant pH is required to support toxicology studies. If the compound is
shown to be hydrolytically unstable in the preliminary study, a more extensiveTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 9

investigation (2 nd tier) may be carried out at different temperatures to better define the hydrolysis kinetics for the compound and identify hydroly tic degradation products.

To better understand the hydrolysis of Tier 1 hydrolysis studies were conducted at 50°C over five days in buffered solutions at pH 4, 7, and 9, and at 37°C for 17 hours in pH 1.2.

EXPERIMENTAL DESIGN
Hydrolysis studies were conducted following the guideline established by the OECD method 111 ( OECD, 2004 ). Buffered solutions (0.05M) were prepared at pH 1.2 (HCl), pH 4 (phosphoric acid), pH 7 (monopotassium phosphate), and pH 9 (boric acid). Final pH adjustments were made using either 1N sodium hydroxide or 1N hydrochloric acid (±0.2 pH units). To reduce the potential for biodegradation on the test compound, the buffered solutions were filter-sterilized through a 0.45- μm nylon membrane (Nalge Company, Rochester, New York). Glassware was sterilized by autoclaving as appropriate. The test compound was added to 10-mL portions of buffered solutions in
20- mL serum vials at a nominal concentration of 3 μg / L. To minimize possible oxidation reactions, test solutions were sparged with argon to reduce the dissolved oxygen concentrations in the reaction mixtures and oxygen concentrations in the headspace of the
serum vials . The serum vials were crimp capped, and labeled with a unique code for
identification . The test solutions were prepared in duplicate. The pH 1.2 solutions were placed in a 37 **°** C incubator and the pH 4, 7 and 9 samples were placed in a 50 **°** C water bath (temperature control 0.5 ± **°** C) . The test solutions were incubated in the dark to minimize possible photochemical reactions

. Analytical Pro cedures
The concentration of the test compound dissolved in an aqueous solution was determined using reverse phase high performance liquid chromatography (HPLC) with mass spectrometry ( MS ) detection

. An Agilent 1 110 series HPLC was used with the gradient

program summarized in T able 1.
The separation of the test substance from possible hydrolysis products was obtained on a Agilent XDB C18 column (4.6 x 50 mm 1.8um). eluted from the column with an approximate retention time of 7.6 minutes. An analytical stock solution
of was prepared in acetonitrile and a series of dilutions were performed in water to achieve the target concentration of 3 μg /L

.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 10

Periodic measurements of the water bath temperature were made with a mercury thermometer to confirm proper temperature of the water bath.

STUDY SPECIFIC PARAMETERS
Data and Reporting
The concentration of the test substance in the pH 4, 7 and 9 test sample s was measured at each sampling point. Hydrolysis half- lives for t he test substance were determined using the calculations described in the OECD method 111 guideline. For hydrolysis reactions that follow pseudo- first order kinetics, a plot of the natural logarithm of the concentration versus time results in a straight l ine (Figure 1). The slope of the line equals –k, the pseudo- first order rate constant. The half -life for the hydrolysis reaction is equal to

t 1/2 = ln 2/k.
The percent degradation was calculated for the pH 1.2 samples at each sampling point to verify that is stable in low pH solutions.

STATISTICS
Descriptive statistics (mean, standard deviation) were used where applicable in this study.

RESULTS AND DISCUSSION
The results for the OECD 111 hydrolysis study for are summarized
in Table 2. No hydrolysis of was noted at pH 1.2 but hydrolysis rates increased
with increasing pH

.
At pH 1.2 there was no loss of the test material from solution over 17 hrs indicating that was hydrolytically stable over this time period. At pH 4 minimal (~7%) loss of from solution was observed also indicating hydrolytic stability at this pH range ). Aq ueous reaction mixtures poised at pH 7 and pH 9 samples exhibited significant loss of over the five days. At pH 7 and 9 the concentration of decreased by 38%
and 100% resulting in a half lives of 7.5 and less than 1 days respectively.

The wate r bath was maintained at 50oC ± 0.0 oC (n=3) during the study based on measurements at the time of sampling.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 11

CONCLUSIONS
A hydrolysis study was conducted for in accordance with the test procedure described in the OECD “Hydrolysis as a Function of pH” guideline (method 111). The showed increasing hydrolysis rates with increasing pH. Percent degradation of the test substance after five days at 50oC was 7% at pH 4, 38% at pH 7, and 100% at pH 9. Corresponding half lives of the test substance were >1 year at pH 4 , 7.5 days at pH 7, and <1 day at pH 9. No detectable degradation of the test substance occurred after 17 hours at pH 1.2 at 37oC.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 12

REFERENCES
OECD (2004). Organisation for Economic Co-operation and Development, (OECD) Guidelines for the Testing of Chemicals . Test Guideline 111: Hydrolysis as a Function of pH. Adopted 13 April 2004.THE DOW CHEMICAL COMPANY STUDY ID: PAGE 13

DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 GUIDELINE

Table 1. Analytical Testing Conditions and Settings
Instrument Components:

Pump: Agilent 1 100 Series; G1312 A; S/N DE 43618194
Autosampler: Agilent 12 0 0 Series; G1367B ; S/N DE 60555149

MS Detector: Agilent 1 100 MSD ; G1946D ; S/N US23412287

Column: Agilent XBD C18; 4.6mm x 50 mm, 1.8 μm
Analytical Conditions : HPLC Eluent (A) = Milli -Q water + 0.1% formic acid
HPLC Eluent (B) = Acetonitrile + 0.1% formic acid

Time (minutes) % Eluent A % Eluent B

0 60 40

3 60 40

6 5 95

10 5 95

12 60 40

16 60 40

Flow = 1.0 mL/min

Detection: MS SIM 359 ion
Retention Time: Approximately 7.6 minutes
Injection Volume: 5 0 μLTHE DOW CHEMICAL COMPANY STUDY ID: PAGE 14

DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 GUIDELINE

Table 2. Hydrolysis Results

a ± 1 standard deviation
b natural logarithm

pH 7

Day Calc Conc (pp b ) % Degradation ln (conc)

0 3.23 ± 0.0 0 - 1.17

1 2.85 ± 0.0 7 12 % 1.05

5 2 .0 1 ± 0. 23 38 % 0 .7 0

pH 7 Half Life = 7 .5 Days pH 1.2 Hour % Degradation

1 0

2 0

17 0

pH 4 Day Concentration ( μg/L ) % Degradation ln b (concentration )

0 3.23 ± 0.00 a – 1.17

1 3.41 ± 0. 21 N one 1.23

5 3.01 ± 0.0 2 7 % 1.10THE DOW CHEMICAL COMPANY STUDY ID: PAGE 15

DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 GUIDELINE

Table 2. Hydrolysis Results (continued)

pH 9 Half Life = < 1 Day pH 9
Day Calc Conc (pp b ) % Degradation ln (conc)

0 3.23 ± 0.0 0 - 1.17

1 0.00 ± 0.0 100 % NA

5 0.00 ± 0. 0 100 % NATHE DOW CHEMICAL COMPANY STUDY ID: PAGE 16

DETERMINATION OF HYDROLYSIS RATE FOLLOWING OECD 111 GUIDELINE

Figure 1. Ln Conc entration vs Time

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40

0 1 2 3 4 5 6Ln Concentration

Time (Days) pH 4

pH 7
pH 9
Linear (pH 4)
Linear (pH 7)
Linear (pH 9)