DIETHYLTOLUENE DIAMINE (DETDA): DETERMINATION OF WATER SOLUBILITY BY THE SHAKE FLASK METHOD

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 471C-144

U.S. EPA Product Properties Test Guideline, OPPTS 830.7840 Water Solubility: Column Elution Method; Shake Flask Method

and

Organisation for Economic C ooperation and Development OECD Guideline 105: Water Solubility

AUTHORS:

William R. Schutt, B.S. Willard B. Nixon, Ph.D.

STUDY INITIATION DATE: January 30, 2012

STUDY COMPLETION DATE: March 19, 2012

SUBMITTED TO:

Albemarle Corporation 451 Florida Street Baton Rouge, LA 70801 

# Wildlife International, Ltd.

8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600

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TABLE OF CONTENTS

Title/Cover Page .............................................................................................................. ....................... 1

Good Laboratory Practice Compliance Statement ................................................................................. 2

Quality Assuran ce Statement ................................................................................................... .............. 3

Report Approval ............................................................................................................... ...................... 4

Table of Contents ............................................................................................................. ...................... 5

Summary ....................................................................................................................... ......................... 7

Introduction .................................................................................................................. .......................... 8

Objective ..................................................................................................................... ........................... 8

Experimental Design ........................................................................................................... ................... 8

Materials and Methods ......................................................................................................... .................. 8 Test Substance ............................................................................................................... ............ 8 Solvents and Reagents ........................................................................................................ ....... 9 Stock and Standards Preparations ............................................................................................. 9 Preliminary Test Procedure ................................................................................................... .. 10 Definitive Test Procedure .................................................................................................... .... 10 Analytical Method ............................................................................................................ ....... 11 Calibration Curve and Limit of Quantitation .......................................................................... 12 Reagent Bla nk Samples ........................................................................................................ ... 12 Example Calculations ......................................................................................................... ..... 12

Results and Discussion ........................................................................................................ ................. 13

Conclusions ................................................................................................................... ....................... 13

Refere nces .................................................................................................................... ........................ 14

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TABLE OF CONTENTS - Continued -

TABLES

Table 1. HPLC Operational Parameters. ......................................................................................... 15

Table 2. Quality Control Samples of DETDA in Reagent Water ................................................... 16

Table 3. Water Solubility of DETDA at 20oC ................................................................................ 17

FIGURES

Figure 1. Analytical method flowchart for the analysis of DETDA in saturated reagent water solutions ...................................................................................... 18

Figure 2. Calibration curve for DETDA .......................................................................................... 19

Figure 3. Representative chromatograms of low- and high-level DETDA calibration standards ....................................................................................................... .. 20

Figure 4. Representative chromatograms of a reagent blank and a 48-hr solubility sample ............ 21

APPENDICES

Appendix 1. Protocol ...................................................................................................................... 22

Appendix 2. Characterization Report .............................................................................................. 32

Appendix 3. Personnel Involved in the Study ................................................................................. 36

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SUMMARY

STUDY TITLE : Diethyltoluene Diamine (DETDA): Determination of Water Solubility by the Shake Flask Method

WILDLIFE INTERNATIONAL , LTD

. PROJECT NUMBER : 471C-144

SPONSOR : Albemarle Corporation 451 Florida Street Baton Rouge, LA 70801

TESTING FACILITY : Wildlife International, Ltd. Easton, Maryland 21601

SPONSOR ’ S REPRESENTATIVE : Marcia L. Hardy, DVM, Ph.D.

LOCATION OF STUDY , RAW
DATA AND A COPY OF THE
FINAL REPORT : Wildlife International Ltd. Easton, Maryland 21601

TEST SUBSTANCE : Diethyltoluene Diamine (DETDA)

TEST DATES : Experimental OECD Start – January 31, 2012 Experimental EPA Start – January 31, 2012 Experimental Termination – February 10, 2012

TEST SYSTEM : HPLC-grade reagent water

S UMMARY : The water solubility of DETDA was determined in reagent water at 20°C using the shake flask method and high performance liquid chromatography with ultraviolet absorbance detection (HPLC/UV). Saturation sub-samples were prepared for analysis by centrifugation followed by dilution of the supernatant into the calibration range of the HPLC/UV methodology with 20:80 (v/v) acetonitrile (ACN): reagent water (H 2 O). The mean water solubility (n = 6) for DETDA at 20 oC was 22.7 mg DETDA/mL +/- 0.44 mg DETDA/mL (CV = 2.0%).

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INTRODUCTION

Tests were conducted to determine the water sol ubility of diethyltoluene diamine (DETDA) at 20  C using the shake flask method. Wildlife International, Ltd. conducted this study for Albemarle Corporation at the Wildlife International, Ltd. anal ytical chemistry facility in Easton, Maryland. The experimental portion of this study was conducted between January 31, 2012 and February 10, 2012. Raw data generated by Wildlife International, Ltd. a nd a copy of the final report are filed under Project

Number 471C-144 in archives located on the Wildlife Inte rnational, Ltd. site. The original signed report will be maintained by Albemarle Corporation, Baton Rouge, Louisiana.

OBJECTIVE The objective of this study was to experimenta lly determine the water solubility of DETDA in reagent water at 20oC using the shake flask method.

EXPERIMENTAL DESIGN The water solubility of DETDA was determined at a temperature of 20  0.1  C. A preliminary

test was conducted to estimate the wa ter solubility. An excess amount of the test substance was shaken in the presence of reagent water at 20  C and sampled after a 24-hour period. The definitive test consisted of shaking an excess amount of test substance with reagent water at an elevated temperature (30  C) for 24, 48, and 72 hours followed by equilibration at 20  C for 24 hours prior to analysis and quantitation of soluble test substance in the aque ous phase of the saturated solution.

MATERIALS AND METHODS The study was conducted according to the procedur es outlined in the protocol, “Diethyltoluene Diamine (DETDA): Determination of Water Solubility by the Shake Flask Method” (Appendix 1). The test was performed based on procedures in the U. S. EPA Product Properties Test Guidelines, OPPTS
830.7840, Water Solubility: Column Elution Method; Shake Flask Method (1) and OECD Guideline for the Testing of Chemicals, 105: Water Solubility (2).

Test Substance

The test substance was received from Albemarl e Corporation on September 21, 2011, and was assigned Wildlife International, Ltd. identificati on number 10232. The test substance was a liquid identified as DETDA; Lot number – 4500051816-011110. A purity of 97.27% was obtained from the

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characterization report provided by the Sponsor (A ppendix 2). The test substance was stored under

ambient conditions.

Solvents and Reagents All solvents were HPLC-grade or higher quality a nd reagents were ACS reagent-grade or better.

Solvent/Reagent Manufacturer/Lot Reagent Water: HPLC-Grade, Burdick & Jackson, Lot Nos. DF291-E and DE958-E (equivalent to ASTM Type II Designation D1193-06 (3)). Acetonitrile (ACN): HPLC-Grade, Burdick & Jackson, Lot Nos. DF154 and DE806

Stock and Standards Preparations A primary stock solution of the test substance was prepared at a nominal concentration of 1.00 mg DETDA/mL by accurately weighing 0.1029 grams of the test substance in a scintillation vial (weight adjusted for 97.27% purity). The test substa nce aliquot was then quantitatively transferred into a 100-mL volumetric flask and was brought to final volume using ACN. The 1.00 mg DETDA/mL (1000 mg DETDA/L) primary stock solution, identified as 10232-001, was used to prepare two sets of standard solutions for the preliminary and definitive test, respectively, in a final solvent composition of 20:80 (v/v) ACN: H 2 O, using the following dilution schemes:

Stock Concentration (mg DETDA/mL) Aliquot ( μ L) Final Volume

(mL) Standard Concentration (mg DETDA/L)

1.00 1.00 1.00 1.00 1.00 1.00 25.0 50.0 100 250 500 1000 10.0 10.0 10.0 10.0 10.0 10.0 2.50 5.00 10.0 25.0 50.0 100

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Stock Concentration (mg DETDA/mL) Aliquot (μL) Final Volume

(mL) Standard Concentration (mg DETDA/L)

1.00 1.00 1.00 1.00 1.00 100 250 500 600 750 5.00 5.00 5.00 5.00 5.00 20.0 50.0 100 120 150

Preliminary Test Procedure A preliminary test was performed to provide an estimate of DETDA water solubility at 20°C. The experiment consisted of shaking an excess amount of DETDA (nominal concentration = 50.0 mg/mL) in reagent water at 20°C and analyzi ng triplicate aliquots of centrifuged supernatant after 24-hours of equilibration. A small quantity of DET DA (1.0007 g) was weighed directly into a clear 40-mL I-Chem glass vial with an analytical balance. Twenty milliliters of reagent water was transferred into the glass vial. The vial was sealed and placed into a shaker bath (Boe kel/Model 290400) maintained at 20°C. After 24 hours, the sample was removed from the bath and triplicate 1.5-mL subsamples were transferred into microcentrifuge tubes and centr ifuged (Eppendorf/MiniSpin Plus) at a setting of 14,500 rpm (~14,100 x g) for 10 minutes. An aliquot of each supernatant was diluted with an appropriate volume of ACN and reagent water to yield a final sample composition of 20:80 (v/v) ACN:H 2 O. Secondary and tertiary dilutions we re performed with 20:80 (v/v) ACN:H 2 O dilution solvent into the calibration range of the HPLC/UV methodology. The final dilutions were then submitted for

determination of the soluble DETDA concentration.

Definitive Test Procedure A definitive test was performed to determ ine the water solubility of DETDA at 20  C. Four solubility samples were prepared at a target nominal concentration of 131 mg/mL. Quadruplicate samples were weighed directly into separate labeled amber 8-mL glass vials on an analytical balance. Five milliliters of reagent water pre-equilibrated at 30  C was added to each of the four glass vials. A volumetric pipet was used to disp ense the water. The vials were sealed with screw caps and Parafilm ®

and placed in an agitating water bath (Boekel/Model 290400) maintained at 30  0.6  C.

After 24 hours of gentle shaking at 30  C, one of the four vials was removed and re-equilibrated at 20°C for 24 hours by placing it in a temperature controlled water bath maintained at 20  0.1°C.

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Following the re-equilibration period at 20°C (and occasional manual agitation), triplicate aliquots (1.0 mL) were removed from the vial and the ali quots were processed for determination of soluble DETDA concentration. An additional sample ali quot (1.0 mL) was taken for determination of supernatant pH. A second and third vial were treated in a similar manner following an initial equilibration period of 48 and 72 hours at 30  0.6  C. The fourth prepared vial (equilibrated at 30  C for longer than 72 hours) was not processed based on the agreement of the results for the 48- and 72-hour samples. Good agreement between consecutive samples indicated that solubility equilibrium had been attained under the test conditions, as solution stirring time no longer had an influence on measured water solubility.

Analytical Method The sample preparation methodology used for the determination of DETDA in reagent water was developed at Wildlife International, Ltd. Triplicate 1.0-mL sub-samples from each saturation sample were centrifuged (Eppendorf/MiniSpin Plus) at a setti ng of 14,500 rpm (14,100 x g) for ten minutes. The supernatant from each centrifuge tube was transferred in to separate amber glass autosampler vials. Using gas-tight syringes and volumetric flasks, an aliquot (50.0-μL) of each sample was diluted in 20:80 (v/v)

ACN:H 2 O into the calibration range of the HPLC/UV methodology. An aliquot of each sample dilution was transferred to an autosampler vial and submitted for analysis of DETDA by HPLC/UV. A method flowchart for the analysis of samples is provided in Figure 1.

Concentrations of DETDA in the samples were determined by an Agilent Series 1100/1200 HPLC system equipped with an Agilent Series 1100 Variable Wavelength Detector set at 300 nm. Chromatographic separations were achieved using a YMC-Pack ODS AM analytical column (150 mm x 4.6 mm, 3-mm particle size). The instrument parameters are summarized in Table 1.

For pH determinations, a 1.0-mL sub-sample from each saturation sample was transferred into separate microcentrifuge tubes and centrifuged at a setting of 14,500 rpm (~ 14,100 x g) for approximately 10 minutes. A 0.5 mL aliquot of each supernatant was transferred to a separate culture

tube and diluted with 0.5 mL of H 2 O using a positive displacement pipettor. The diluted samples were then equilibrated at 20°C in a water bath for a mi nimum of 30 minutes prior to pH measurement using a calibrated pH meter (Thermo Orion/4 Star Plus).

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Calibration Curve and Limit of Quantitation Five calibration standards of DETDA in 20:80 (v/v) ACN: H 2 O, ranging in concentration from 20.0 to 150 mg DETDA/L, were analyzed concurren tly with the definitive solubility samples. The calibration set was injected at the beginning and e nd of the analytical sequence with one calibration standard injected after a maximum of every five test samples. Linear regression equations were generated for the analytical sequence using the summed/tota l peak area response for retention times between approximately 7.6 and 7.7 minutes versus the resp ective DETDA concentrations of the calibration

standards. The concentration of DETDA in the sam ples was determined by substituting the total peak area responses for retention times between approximately 7.6 and 7.7 minutes of the samples into the applicable linear regression equation. The calibra tion curve for DETDA is presented in Figure 2. Representative chromatograms of low- and high-le vel DETDA calibration standards are presented in Figure 3.

The limit of quantitation (LOQ) was arbitrarily de fined using the low-level calibration standard equivalent definition. For this analysis, the met hod LOQ for the test substance in reagent water was defined as 4.00 mg DETDA/mL, calculated as the product of the concentration of the lowest-level calibration standard (20.0 mg DETDA/L) and the method dilution factor for the reagent blank samples (200) divided by a unit conversion factor (1000 mL/L).

Reagent Blank Samples Three reagent blank quality control (QC) samples in reagent water were processed in the same manner and analyzed concurrently with definitiv e trial solubility samples to determine possible

interferences. No interferences were observed at or above the LOQ during sample analyses (Table 2). A chromatogram of a representative reagent blank sample is presented in Figure 4.

Example Calculations The measured concentration of the test substance in sample number 471C-144-2-A was

calculated as follows:

Test Substance (mg DETDA/mL) in sample = Total Peak Area - (Y-intercept) Slope x Dilution Factor / Unit Conversion Factor

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Total Peak Area = 3708.44946 Sample (Initial) Volume (V i ) = 0.0500 mL Y-intercept = -453.57442 Final Volume (V f ) = 10.0 mL Slope = 36.52221 Dilution Factor (V f /V i ) = (10.0/0.0500) = 200 Unit Conversion Factor = 1000 mL/L

Test Substance (mg DETDA/mL) in sample = 3708.44946 - (-453.57442) 36.52221 x 200 / 1000

Test Substance (mg DETDA/mL) in sample = 22.8

Calculations performed using Microsoft ® Excel 2000 in the full precision mode. Manual calculations may differ slightly.

RESULTS AND DISCUSSION The water solubility of DETDA was estimated from a preliminary trial that consisted of gentle shaking of an excess of the test substance in reagen t water at 20°C for 24 hours. The mean measured test substance solubility under the test condition was 21.5 mg DETDA/mL +/- 0.25 mg DETDA/mL (CV = 1.1%, N = 3). Based on this finding, the target nominal concentration for DETDA in the definitive test was set at a concentration of 131 mg/mL.

The pH of the test system water and samples was evaluated concurrent with the definitive solubility test. The pH of the test system (reagen t) water at 20°C was 8.50. The measured pH from the diluted supernatant of reagent water saturated with DETDA at 19.6, 23.1, and 22.3 mg DETDA/mL at 20°C was 9.45, 9.14, and 9.18 for saturation samples shaken and equilibrated at 30  C for 24, 48, and 72 hours prior to 24-hour equilibration at 20  C, respectively.

Mean measured water-soluble DETDA concentr ations for saturation samples shaken and equilibrated at 30°C for 48 and 72 hours prior to 24-hour equilibration at 20°C were well within the 15% difference criterion of the protocol (3.3%). The mean water solubility (n = 6) for DETDA at 20 oC was 22.7 mg DETDA/mL +/- 0.44 mg DETDA/mL (CV = 2.0%) (Table 3). A representative chromatogram of a DETDA water solubility sample is presented in Figure 5.

CONCLUSIONS The mean water solubility (n = 6) for DETDA at 20 oC was 22.7 mg DETDA/mL +/- 0.44 mg DETDA/mL (CV = 2.0%).

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REFERENCES

1. U.S. EPA Product Properties Test Guidelines. 1998. OPPTS 830.7840. Water Solubility: Column Elution Method; Shake Flask Method

.

2. Organisation for Economic Cooperation and Development. 1995. Guideline for Testing of Chemicals, 105: Water Solubility

.

3. American Society for Testing and Materials. 2006. Standard Specification for Reagent Water. D1193-06, ASTM Section II Water and Envi ronmental Technology, Vol. 11.01: 45-47.

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Table 1

HPLC Operational Parameters

INSTRUMENT: Agilent Series 1100/1200 High Performance Liquid Chromatograph (HPLC) with an Agilent Series 1100 Variable Wavelength Detector

ANALYTICAL COLUMN: YMC-PACK ODS-AM C18 (150 mm x 4.6 mm, 3-  m particle size)

STOP TIME: 13.00 minutes

FLOW RATE: 1.000 mL/minute

OVEN TEMPERATURE: 40.0°C

MOBILE PHASE: Solvent A (80.0%): HPLC-grade Reagent Water Solvent B (20.0%): Acetonitrile

GRADIENT ELUTION PROFILE: TIME (min.) SOLVENT A (%) SOLVENT B (%) FLOW (mL/min.) 0.01 80.0 20.0 1.000 2.00 80.0 20.0 1.000 8.00 5.0 95.0 1.000 10.00 5.0 95.0 1.000 10.01 80.0 20.0 1.000 13.00 80.0 20.0 1.000

INJECTION VOLUME: 50.0 μL

DETDA RETENTION TIME RANGE: Peak area 1: 7.6 minutes Peak area 2: 7.7 minutes

UV WAVELENGTH: 300 nm

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Table 2

Quality Control Samples of DETDA in Reagent Water

Sample Number (471C-144-) Sample Type Sampling Interval

(hrs) Concentrations of DETDA Percent Recovered 2 Mean Percent Recovered 2 Fortified (mg DEDTA/mL) Measured (mg DETDA/mL) 2

REB-1 REB-2 REB-3 Reagent Blank Reagent Blank Reagent Blank 24 48 72 0.0 0.0 0.0 < LOQ 1 < LOQ 1 < LOQ 1 --

--

-- --

1 The Limit of Quantitation (LOQ) for determination of DETDA in reagent water (4.00 mg DETDA/mL ) was calculated as the product o f the lowest-level calibration standard (20.0 mg DETDA/L) and the dilution factor of th e reagent blank samples (200) divided by a unit conversion factor (1000 mL/L).

2 Results were generated using Microsoft ® Excel 2000 in full precision mode. Manual calculations may differ slightly.

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Table 3

Water Solubility of DETDA at 20oC

Sample Number (471C-144-) Time at 30oC (hrs) Concentrations of DETDA Nominal (mg DETDA/mL) Measured (mg DETDA/mL) 2 Mean Measured +/- SD (mg DETDA/mL) 1,2 % Difference

1-A 1-B 1-C

2-A 2-B 2-C

3-A 3-B 3-C 24 24 24

48 48 48

72 72 72 131 131 131

131 131 131

131 131 131 18.6 18.8 21.5

22.8 23.2 23.2

22.2 22.3 22.4 19.6 +/- 1.6 (CV = 8.3%)

23.1 +/- 0.23 (CV = 1.0%)

22.3 +/- 0.12 (CV = 0.55%) 16% 3

3.3% 4

Overall Mean 5 = SD = CV =

N = 22.7 0.44 2.0%

6

1 SD = Standard Deviation; CV = Coefficient of Variation; N = Number of Measurements.

2 Results were generated using Microsoft ® Excel 2000 in full precision mode. Manual calculations may differ slightly.
3 %Difference = [absolute value(mean m easured result 24hrs - mean measured result 48hrs)] / [0.5 x (mean measured result 24hrs + mean measured result 48hrs)] x 100.

4 %Difference = [absolute value(mean m easured result 48hrs - mean measured result 72hrs)] / [0.5 x (mean measured result 48hrs + mean measured result 72hrs)] x 100.

5 Overall mean calculated for 48 and 72 hours.

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Using glass pasteur pipets, transfer triplicate approximately 1.0-mL aliquot s of each solubility saturation sample into labeled microcentrifuge tubes, appending solubility sample IDs with 'A', 'B' and '-C'). Seal tubes.



Centrifuge tubes at a setting of 14500 rpm for 10 minutes.



Transfer each supernatant into labeled amber glass autosa mpler vials. Prepare a primary dilution by transferring 50.0 μL of each sample into properly labeled 10.0-mL volumetric flasks partially filled with 20: 80 ACN: H 2 O (v/v) dilution solvent. Bring samples to final volume with dilution solvent. Cap and invert several times to mix. Prepare a regent blank in the same manner using reagent water.



If necessary, perform additional serial dilutions in 20: 80 ACN: H 2 O (v/v) dilution solvent using the appropriate

glassware. Transfer an aliquot of each final diluti on into an autosampler vial, cap, and submit for HPLC/UV analysis. Place remainder of original aqueous sample s upernatant and sample dilutions in refrigerated storage.

Figure 1 . Analytical method flowchart for the analysis of DETDA in saturated reagent water solutions.

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### Calibration Curve (Summed Peaks)

y = 36.52221x - 453.57442

R

2 = 0.99817

0 1000 2000 3000 4000 5000 6000

0.0 25.0 50.0 75.0 100.0 125.0 150.0

DETDA Concentration (mg a.i./L)Total Peak Area (300 nm)

Figure 2. A representative calibration curve for DETDA.

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min 0 2 4 6 8 10 12 mAU

0 200 400 600 800 1000 1200 VWD1 A, Wavelength=300 nm (X:\HPLC13\1\DATA\DETDA_WS003\DETDA_WS003 2012-02-09 15-00-37\002-0201.D)7.6227.754

(A)

min 0 2 4 6 8 10 12 mAU

0 200 400 600 800 1000 1200 VWD1 A, Wavelength=300 nm (X:\HPLC13\1\DATA\DETDA_WS003\DETDA_WS003 2012-02-09 15-00-37\006-0601.D)7.6197.751

(B)

Figure 3. Representative chromatograms of low-level a nd high-level DETDA calibration standards.

(A): Low-level calibration standard (20.0 mg DETDA/L) (B): High-level calibration standard (150 mg DETDA/L)

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min 0 2 4 6 8 10 12 mAU

0 200 400 600 800 1000 1200 VWD1 A, Wavelength=300 nm (X:\HPLC13\1\DATA\DETDA_WS003\DETDA_WS003 2012-02-09 15-00-37\007-0701.D)

(A)

min 0 2 4 6 8 10 12 mAU

0 200 400 600 800 1000 1200 VWD1 A, Wavelength=300 nm (X:\HPLC13\1\DATA\DETDA_WS003\DETDA_WS003 2012-02-09 15-00-37\008-0801.D)7.6207.753

(B)

Figure 4. Representative chromatograms of a reagent blank and a 48-hr solubility sample.

(A): REB-2 (20:80 (v/v) ACN:H 2 O; Dilution factor = 200). The arrows represent the approximate retention times of the peak areas of DETDA test substance. (B): 2-A (48-hour solubility sample. Dilution factor = 200)

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Appendix 1

Protocol

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Appendix 2

Characterization Report

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Appendix 3

Personnel Involved in the Study

The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study:

1. Willard B. Nixon, Ph.D., Director of Chemistry

2. Raymond L. Van Hoven, Ph.D., Scientist

3. William R. Schutt, B.S., Chemist

4. John R. Murrell, B.S., Senior Chemist

5. David Weiss, M.S., Chemist