FINAL REPORT

Study Title: An Analytical Method Validation, Compatibility, 

and Stability Study of Acetaldehyde in Deionized Water

Laboratory Project ID: WIL-876002

Author: Scott D. Freer, BA

Test Guideline: Not Applicable

Study Completion Date: 01 March 2013

Performing Analytical 

Laboratory:

WIL Research 

1407 George Road

Ashland, OH 44805-8946

Sponsor: Acetaldehyde Working Group of the 

Vinyl Acetate Council, Inc.

1250 Connecticut Ave, NW

Suite 700

Washington, DC 20036

Total Number of Pages: 60

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WIL-876002 Acetaldehyde 

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc. 

COMPLIANCE STATEMENT 

The following is a detailed description of all differences between the practices used in the 

study, WIL-876002, and those required by the United States FDA GLP Regulations 

(21 CFR Part 58), 5 October 1987; United States EPA GLP Standards 40 CFR Part 792 

(18 September 1989); the OECD Principles of GLP [C(97) 186iFinall (26 November 

1997): 

0 On 14 December 2011, 19 December 2011, and 22 December 2011 the actual 

weights/masses of the 0.5 M citric acid and DNPH derivatizing agent were 

inadvertently not recorded at the time of preparation. This exception to the GLP 

standards did not affect the quality or integrity of the study because all 

chromatography was historically consistent and all data met acceptance criteria. 

A Certificate of Analysis for the test substance was provided by the supplier 

(presented in Appendix B). 

0 

Typed name: Scott D. Freer, BA 

Typed name of Laboratory: WIL Research 

Sponsor: 

Signature Date 

Typed name: 

Typed name of Company: 

Submitter: 

Signature Date 

Typed name: 

Typed name of Company: 

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TABLE OF CONTENTS

Page

Compliance Statement...............................................................................2

Table of Contents.......................................................................................3

Index of Figures..........................................................................................5

Index of Tables...........................................................................................5

Index of Appendices...................................................................................6

1. Summary.....................................................................................................7

2. Introduction................................................................................................8

2.1. Key Study Dates ..........................................................................................9

2.2. WIL Research Key Study Personnel............................................................9

3. Experimental Procedures - Materials and Methods.............................10

3.1. Test Substance and Vehicle .......................................................................10

3.1.1. Test Substance Identification.....................................................................10

3.1.2. Vehicle Identification.................................................................................10

3.2. Drinking Water Bottles..............................................................................10

3.3. High Performance Liquid Chromatography ..............................................11

3.4. Preparation of Mobile Phase......................................................................11

3.5. Preparation of 0.5 M Citrate Acid..............................................................11

3.6. Preparation of 0.5 M Sodium Citrate.........................................................11

3.7. Preparation of Diluent................................................................................11

3.8. Preparation of Derivatizing Agent Solution ..............................................12

3.9. Preparation of Calibration Stock Solutions................................................12

3.10. Preparation of Calibration Standards.........................................................12

3.11. Preparation of Quality Control Stock Solution..........................................13

3.12. Preparation of Quality Control Samples....................................................13

3.13. Formulation Sample Preparation ...............................................................14

3.14. Formulation Sample Collection and Processing........................................14

3.15. Concentration Quantitation........................................................................15

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Page

3.16. WIL Research Computer Systems.............................................................15

3.16.1. Reporting and Ancillary Systems ..............................................................15

4. Results and Discussion.............................................................................16

4.1. Specificity/Selectivity................................................................................19

4.2. Assay Validation: Calibration Reproducibility.........................................19

4.3. Assay Validation: Precision and Accuracy...............................................20

4.4. Assay Ruggedness .....................................................................................20

4.5. Assay Acceptability ...................................................................................20

4.6. Test Substance Stability in Calibration Standards.....................................21

4.7. Test Substance Stability in Processed Samples.........................................21

4.8. Test Substance Time-Zero Concentration Assessment of 

Formulations..............................................................................................21

4.9. Test Substance Stability of Formulations in Glass Containers..................22

4.10. Test Substance Compatibility of Formulations in Drinking Water 

Bottles........................................................................................................23

5. Conclusions...............................................................................................24

6. Report Review and Approval .................................................................25

7. Quality Assurance Statement..................................................................26

8. Data Retention..........................................................................................27

9. Abbreviations...........................................................................................28

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INDEX OF FIGURES

Page

1. Representative Chromatogram of a 

10.0 μg Acetaldehyde/mL Calibration Standard .......................................16

2. Representative Chromatogram of a Processed 

5.00 mg Acetaldehyde/mL Quality Control Sample..................................17

3. Representative Chromatogram of a Processed 

5.00 mg Acetaldehyde/mL Formulation Sample.......................................17

4. Chromatogram of a Processed Vehicle Blank Sample ..............................18

5. Chromatogram of a Processed Diluent Sample .........................................18

INDEX OF TABLES

1. Back-Calculated Concentrations of the Validation Calibration 

Standards....................................................................................................30

2. Calculated Concentrations of the Validation Quality Control 

Samples......................................................................................................31

3. Minimum 5-Day Room Temperature Stability Analysis of the

14 December 2011 Calibration Standards and Processed Quality

Control Samples.........................................................................................32

4. Concentration Assessment of the 19 December 2011 Formulations.........33

5. 24-Hour Room Temperature Stability Assessment of the 

19 December 2011 Formulations (Glass Container) .................................34

6. 48-Hour Room Temperature Stability Assessment of the 

19 December 2011 Formulations (Glass Container) .................................35

7. 72-Hour Room Temperature Stability Assessment of the 

19 December 2011 Formulations (Glass Container) .................................36

8. 24-Hour Room Temperature Stability Assessment of the 

19 December 2011 Formulations (Drinking Bottles) ................................37

9. 48-Hour Room Temperature Stability Assessment of the 

19 December 2011 Formulations (Drinking Bottles) ................................38

10. 72-Hour Room Temperature Stability Assessment of the 

19 December 2011 Formulations (Drinking Bottles) ................................39

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INDEX OF APPENDICES

Page

A. Study Protocol and Deviations...................................................................40

B. Certificate of Analysis ...............................................................................59

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1. SUMMARY

A high performance liquid chromatography method using ultraviolet absorbance 

detection at a wavelength of 360 nm for the determination of acetaldehyde concentration 

in deionized (DI) water formulations containing test substance ranging in concentration 

from 1.00 to 5.00 mg/mL was validated in this study. Also in this study, test substance

stability was assessed in calibration standards and processed quality control (QC) 

samples stored at room temperature for a minimum of 5 days.

In addition, test substance concentration acceptability and, following 24, 48, and 72 hours 

of room temperature storage in glass containers, stability were assessed in DI water 

formulations prepared at target concentrations of 1 and 5 mg acetaldehyde/mL. In

addition, test substance formulation compatibility with drinking water bottles following 

24, 48, and 72 hours of room temperature storage was assessed in DI water formulations 

prepared at target concentrations of 1.00 and 5.00 mg acetaldehyde/mL.

The acetaldehyde assay procedure was validated in this study with 3 validation sessions. 

Quantitation was performed using calibration standards ranging in test substance

concentration from 10.0 to 50.0 μg/mL. The mean back-calculated standard 

concentrations had inter-session variability ranging from 0.54% to 2.3% relative standard 

deviation (RSD) and percent relative error (%RE) ranging from -0.30% to 0.16%, which 

met the protocol-specified acceptance criteria for calibration standards, i.e., RSD ≤10% 

and %RE within ± 10% (except at the lowest level where RSD ≤15% and %RE within 

± 15% were acceptable). Assay precision and accuracy were verified by the analysis of 

QC samples prepared at 1.00, 3.00, and 5.00 mg acetaldehyde/mL. The mean calculated 

QC concentrations had inter-session variability (precision) ranging from 5.7% to 

7.9% RSD and %RE (accuracy) ranging from -5.0% to -3.4%. The results met the 

protocol-specified acceptance criteria for precision and accuracy, i.e., RSD ≤10% and 

%RE within ± 10%.

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Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

The test substance in calibration standards and processed QC samples stored at room 

temperature for a minimum of 5 days met the protocol-specified acceptance criteria for 

stability, i.e., the post-storage concentration was not <90% of the pre-storage value.

The results of the test substance concentration acceptability assessment in DI water 

formulations prepared at target concentrations of 1 and 5 mg acetaldehyde/mL met the 

protocol-specified acceptance criteria for concentration acceptability, i.e., the mean 

concentration was 90% to 110% of the target concentration. Assessment of test 

substance stability in glass containers following 24, 48, and 72 hours of room 

temperature storage in DI water formulations prepared at target concentrations of 1 and 

5 mg acetaldehyde/mL met the previously stated protocol-specified requirement for 

stability with the following exception. The low group (1 mg/mL) formulation prepared 

on 19 December 2011 and stored at room temperature for 72 hours was 88.8% of the 

time-zero concentration which did not meet the stated criteria for stability.

The results of the test substance compatibility assessment with drinking water bottles 

following 24, 48, and 72 hours of room temperature storage in DI water formulations 

prepared at target concentrations of 1 and 5 mg acetaldehyde/mL met the 

protocol-specified acceptance criteria for compatibility acceptability, i.e., the post-storage 

assay concentration was not <95% of the post-storage concentration of the formulation 

stored in glass containers.

2. INTRODUCTION

This report provides a detailed description and validation of a high performance liquid 

chromatography (HPLC) method using ultraviolet (UV) absorbance detection at a 

wavelength of 360 nm for the determination of acetaldehyde concentration in 

deionized (DI) water formulations and test substance ranging in concentration from 

1.00 to 5.00 mg/mL. Assay specificity/selectivity, calibration reproducibility, precision, 

accuracy, ruggedness, and test substance stability in calibration standards and processed 

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QC samples stored at room temperature for a minimum of 5 days were established and 

verified.

Also in this study, DI water formulations prepared at target concentrations of 1 to 

5 mg acetaldehyde/mL were analyzed to assess test substance concentration acceptability 

and, following 24, 48, and 72 hours of room temperature storage in glass containers, 

stability. In addition, DI water formulations prepared at target concentrations of 1 to 

5 mg acetaldehyde/mL were analyzed to assess test substance compatibility with drinking 

water bottles following 24, 48, and 72 hours of room temperature storage.

The study protocol is presented in Appendix A.

A list of abbreviations potentially used in this report is presented in 

Section 9. (Abbreviations).

2.1. KEY STUDY DATES

Date(s) Event(s)

07 November 2011 ................................. Study Initiation Date

14 December 2011..................................Experimental start (starting) date 

(first date of analysis)

23 December 2011..................................Experimental termination (completion) date 

(last date of analysis)

2.2. WIL RESEARCH KEY STUDY PERSONNEL

Robert E. Boes Jr., MS Associate Research Chemist, Analytical Chemistry

Dennis J. Hoobler, BS Training Coordinator, Analytical Chemistry

Phong X. Pham, BS Chemist II, Analytical Chemistry

Gregory A. Hawks, AS Group Supervisor, Reporting & Technical Support 

Services

Melissa A. Hull, BS WIL Research - US Manager, Computerized Systems 

Validation

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Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

3. EXPERIMENTAL PROCEDURES - MATERIALS AND METHODS

3.1. TEST SUBSTANCE AND VEHICLE

3.1.1. TEST SUBSTANCE IDENTIFICATION

The test substance, acetaldehyde, was received from Sigma-Aldrich, Milwaukee, WI on 

04 October 2011 as follows:

Identification Quantity Received Physical Description

Acetaldehyde

Lot no. SHBB4508V

[WIL ID no. 110159]

20 Containers

Gross volume: 20 L Colorless liquid

Documentation regarding the purity and stability of the test substance is on file with the 

Sponsor and WIL Research. A Certificate of Analysis for the test substance was 

provided by the vendor and is presented in Appendix B. The purity of the test substance

was ≥99.5%. The test substance was stored refrigerated, protected from light, under 

nitrogen cap and was considered stable under these conditions.

3.1.2. VEHICLE IDENTIFICATION

The vehicle used in preparation of the test substance formulations was DI water. 

3.2. DRINKING WATER BOTTLES

The drinking water bottles used to determine compatibility with the DI water 

formulations were received 30 November 2011 as follows:

● 6 empty 400-mL polycarbonate bottles with silicone rings (list no. ACBT0402, 

received from Tecniplast USA Inc., West Chester, PA)

● 6 S/S caps with 2 balls (list no. ACCPS50, received from Tecniplast USA Inc.)

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3.3. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Instrument: Agilent 1100 liquid chromatograph equipped with a 

variable wavelength detector, autosampler, and Dionex 

Chromeleon®software, or equivalent system

Column: Zorbax ODS column, 4.6 mm × 250 mm., 

5-μm particle-size

Mobile Phase A: 60:40 (v/v) Acetonitrile (ACN):DI water

Flow Rate: 1.50 mL/minute

Column Temperature: 25.0°C

Detector: UV at 360 nm

Injection Volume: 20.0 μL

Retention Time: Approximately 5.2 minutes for acetaldehyde

Run Time: 15 minutes

3.4. PREPARATION OF MOBILE PHASE

Mobile phase was prepared by combining ACN and DI water in a 60:40 (v/v) ratio.

3.5. PREPARATION OF 0.5 M CITRATE ACID

A 0.5 M citrate acid solution was prepared by dissolving 96.05 g of citric acid in 

1000 mL of DI water.

3.6. PREPARATION OF 0.5 M SODIUM CITRATE

A 0.5 M sodium citrate solution was prepared by dissolving 147.05 g of sodium citrate in 

1000 mL of DI water. 

3.7. PREPARATION OF DILUENT

The diluent was prepared by mixing 800 mL of 0.5 M citric acid with 200 mL of 

0.5 M sodium citrate. 

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3.8. PREPARATION OF DERIVATIZING AGENT SOLUTION

The derivatizing agent, 2,4-dinitrophenylhydrazine (DNPH), was prepared at a 

concentration of 3.00 mg/mL by weighing approximately 0.107 g of 2,4-DNPH 

(30% water) in a tared glass weigh boat. The derivatizing agent was transferred to a 

10-mL volumetric flask with rinses of ACN. The preparation was mixed as necessary to 

achieve complete dissolution of the derivatizing agent. Additional ACN was added to 

obtain the desired concentration, and the solution was thoroughly mixed.

3.9. PREPARATION OF CALIBRATION STOCK SOLUTIONS

The calibration stock solution was prepared at a concentration of 5.00 mg 

acetaldehyde/mL as follows. Approximately 0.05032 g of acetaldehyde (WIL ID

no. 110159; density 0.788g/mL, no correction for purity) was accurately weighed into a 

tared glass container containing 10.0 mL of DI water. Additional DI water was added to 

achieve the desired concentration, and the solution was mixed with vortex action and/or 

gentle swirling.

A secondary calibration standard stock solution was prepared at a concentration of 

0.500 mg acetaldehyde/mL by diluting an aliquot of the calibration stock solution 

10-fold with DI water.

3.10. PREPARATION OF CALIBRATION STANDARDS

Calibration standards at concentrations ranging from 10.0 to 50.0 μg acetaldehyde/mL 

were prepared by diluting aliquots of the secondary calibration stock solution with diluent 

in amber autosampler vials. A 450-μL aliquot of the derivatizing agent solution was 

added to each vial, and the samples were placed in a heated orbital shaker for at least 

1 hour at approximately 40°C at 75 rpm and 100 rpm. Calibration standards were 

prepared in triplicate at each concentration for the validation sessions; at least single 

calibration standards were prepared at each concentration for routine analyses.

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3.11. PREPARATION OF QUALITY CONTROL STOCK SOLUTION

A QC stock solution was prepared at a concentration of 5.00 mg acetaldehyde/mL as 

follows. Approximately 0.05032 g of acetaldehyde (WIL ID no. 110159; density 

of 0.788g/mL, no correction for purity) was accurately weighed into a tared glass 

container containing 10.0 mL of DI water. Additional DI water was added to achieve the 

desired concentration and the solution was mixed with vortex action and/or gentle 

swirling.

3.12. PREPARATION OF QUALITY CONTROL SAMPLES

As detailed in the following table, QC samples were prepared to simulate the 

processing of formulations at acetaldehyde concentrations of 1.00, 3.00, and 

5.00 mg acetaldehyde/mL (nominal QC concentration) by combining aliquots of the QC 

stock solution, vehicle (DI water), and diluent in glass tubes. Portions of the QC samples 

were further diluted with diluent in autosampler vials as needed. A 450-μL aliquot of the 

derivatizing agent solution was added to each vial, and the samples were placed in a 

heated orbital shaker for at least 1 hour at approximately 40°C at 75 rpm and 100 rpm. 

The QC samples were prepared in triplicate at each concentration; a single vehicle blank 

sample was prepared.

QC 

Level

Nominal 

QC Conc.

(mg/mL)

Vehicle 

Volume

(mL)

QC Stock 

Conc.

(mg/mL)

QC Stock 

Volume

(mL)

Diluent 

Volume

(mL)

Secondary

Dilution

Theoretical 

Final Conc.

(μg/mL)

Blank 0.00 1.00 NA NA 9.00 5-fold 0.00

QC1 1.00 1.00 5.00 0.200 8.80 5-fold 20.0

QC2 3.00 1.00 5.00 0.600 28.40 5-fold 20.0

QC3 5.00 1.00 5.00 1.00 48.0 5-fold 20.0

NA = Not applicable

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Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

3.13. FORMULATION SAMPLE PREPARATION 

Duplicate formulation samples were prepared at a concentration of 5 mg 

acetaldehyde/mL as follows. Approximately 2.05 g of acetaldehyde (WIL ID

no. 110159, density 0.788 g/mL, no correction for purity) was accurately weighed into a 

tared glass container or a tared drinking water bottle containing approximately 50.0 and 

100 mL of DI water, respectively. The containers were brought to a final volume of 

400 mL and thoroughly mixed. Additional DI water was added to achieve the desired 

final concentration.

Duplicate formulation samples were prepared at a concentration of 

1 mg acetaldehyde/mL by diluted the 5 mg acetaldehyde/mL formulations 5-fold with DI 

water. The preparations were thoroughly mixed.

3.14. FORMULATION SAMPLE COLLECTION AND PROCESSING

Quadruplicate samples were collected from each formulation using a syringe and dosing 

cannula and placed in glass tubes. Two samples from each quadruplicate set were 

processed for analysis, and the remaining 2 samples (back-up samples) were stored at 

room temperature and, if not needed for analysis, discarded after receipt of the Study

Director’s approval of the analytical results. As detailed in the following table, 

formulation samples were processed by adding diluent and mixing with vortex action. 

A portion of each formulation sample was further diluted with diluent in autosampler 

vials as necessary to achieve a theoretical final test substance concentration within the 

calibration concentration range. A 450-μL aliquot of the derivatizing agent solution was 

added to each sample, and the samples were placed in a heated orbital shaker for 1 hour 

at approximately 40°C at 100 rpm.

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Group

Target Test Substance

Concentration

(mg/mL)

Sample 

Amount

(mL)

Diluent 

Volume

(mL)

Secondary

Dilution

Theoretical Final 

Concentration

(μg/mL)

Low 1 1.0 4.00 10-fold 20.0

High 5 1.0 19.0 10-fold 25.0

NA = Not applicable

3.15. CONCENTRATION QUANTITATION

Single injections were made of each processed calibration, QC, and formulation sample. 

A calibration curve was constructed for each set of analyses. The acetaldehyde peak 

areas (y) and the theoretical concentrations (x) of the calibration standards were fit with 

least-squares regression analysis to the linear function with 1/x weighting:

y = ax + b

Concentrations were calculated from the results of the regression analysis using Dionex 

Chromeleon®software. The concentration data were transferred to a Microsoft Excel®

spreadsheet, where appropriate summary statistics, i.e., mean, standard deviation (SD), 

relative standard deviation (RSD), percent relative error (%RE), and concentration as a 

percent of target, were calculated and presented in tabular form. The concentrations of 

the formulation and QC samples were calculated by applying any necessary 

multiplication factors to correct for dilution and/or unit conversions.

3.16. WIL RESEARCH COMPUTER SYSTEMS

3.16.1. REPORTING AND ANCILLARY SYSTEMS

Program/System Description

Archive Management System 

(AMS)

In-house developed application for storage, maintenance, and 

retrieval of information for archived materials (e.g., lab 

books, study data, wet tissues, slides, etc.) 

InSight®Publisher Electronic publishing system (output is Adobe Acrobat, PDF)

Master Schedule Maintains the master schedule for the company. 

Microsoft Office®2007 Used in conjunction with the publishing software to generate 

study reports.

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4. RESULTS AND DISCUSSION

Under the described chromatographic conditions, the retention time of the test substance

was approximately 5.2 minutes. Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5 are 

typical chromatograms of a calibration standard, a processed QC sample, a processed 

formulation sample, a processed vehicle blank sample, and a processed diluent sample,

respectively. The total analysis time required for each run was 15 minutes.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 15.0

-500

1,000

2,000

3,500 LC7-876002H #10 Run # 301 876002-90-1 UV_VIS_1

m AU

m in

1 - Peak 1 - 5.153

WVL:360 nm

Figure 1: Representative Chromatogram of a 10.0 μg Acetaldehyde/mL Calibration 

Standard

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0.0 2.0 4.0 6.0 8.0 10.0 12.0 15.0

-500

1,000

2,000

3,500 LC7-876002H #34 Run # 325 876002-93-8 UV_VIS_1

m AU

m in

1 - Peak 1 - 5.163

WVL:360 nm

Figure 2: Representative Chromatogram of a Processed 5.00 mg Acetaldehyde/mL

Quality Control Sample

0.0 2.0 4.0 6.0 8.0 10.0 12.0 15.0

-500

1,000

2,000

3,500 LC7-876002H #59 Run # 350 876002-98-3 UV_VIS_1

m AU

m in

1 - Peak 1 - 5.169

WVL:360 nm

Figure 3: Representative Chromatogram of a Processed 5.00 mg Acetaldehyde/mL

Formulation Sample

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0.0 2.0 4.0 6.0 8.0 10.0 12.0 15.0

-15

20

40

60

80

100 LC7 -8 76002 H #27 R un # 318 876002-93-1 UV_VIS_1

m AU

m in

1 - Peak 1 - 5.049

WVL:360 nm

Figure 4: Chromatogram of a Processed Vehicle Blank Sample

0.0 2.0 4.0 6.0 8.0 10.0 12.0 15.0

-1.0

2.0

4.0

6.0

8.0

10.0

LC7-876002H #25 Run # 316 Dil 876002-82-1 UV_VIS_1

m AU

m in

WVL:360 nm

Figure 5: Chromatogram of a Processed Diluent Sample

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4.1. SPECIFICITY/SELECTIVITY

As shown in Figure 4, a small interfering peak (approximately 5.0 minutes) was observed 

in processed vehicle samples near the retention time of the test substance (approximately 

5.2 minutes). The interference did not significantly affect the quantitation of the test 

substance-containing formulations. As shown in Figure 5, analysis of processed 

diluent samples revealed that there were no significant peaks (with signal-to-noise 

ratio [S/N] >10) at or near the retention time for the test substance (approximately 

5.2 minutes).

4.2. ASSAY VALIDATION: CALIBRATION REPRODUCIBILITY

During each of the 3 method validation sessions, triplicate calibration standards at 

5 concentrations were prepared and analyzed as described previously. Single injections 

were made of each calibration standard. The resulting acetaldehyde peak area versus 

theoretical acetaldehyde concentration data were fit to the linear function using 

least-squares regression analysis. The results of the regression analyses were used to 

back-calculate the corresponding concentrations from the peak area data. As per protocol 

criteria, the reproducibility of the calibration curve data was considered valid when 1) the 

inter-session variability, expressed as RSD, of the back-calculated concentrations at each 

calibration level was ≤10%, except at the lowest calibration level where ≤15% was 

acceptable; and 2) the mean back-calculated concentrations at each calibration level were 

within ± 10% of the theoretical values (percent relative error [%RE] within ± 10%), 

except at the lowest calibration level where %RE within ± 15% was acceptable.

The back-calculated concentrations and the associated intra- and inter-session statistics 

for the acetaldehyde assay calibration standards are summarized in Table 1. The 

inter-session variability (RSD) of the back-calculated concentrations ranged from 

0.54% to 2.3% RSD. The inter-session mean concentrations had %RE values ranging 

from -0.30% to 0.16%. Based on the stated criteria, the reproducibility of the calibration 

data was acceptable. 

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4.3. ASSAY VALIDATION: PRECISION AND ACCURACY

During each of 3 validation sessions, triplicate QC samples at 3 concentrations were 

prepared and analyzed as described previously. Single injections were made of each 

processed QC sample. The results of the regression analyses were used to calculate the 

corresponding concentrations from the QC peak area data. The variability (RSD) of the 

calculated QC concentration data was used as a measure of assay precision, and the 

difference between theoretical and the calculated mean QC concentrations (%RE) was 

used as a measure of assay accuracy. According to protocol criteria, the precision of the 

method was considered acceptable when the inter-session RSD of the calculated 

concentrations at each QC level was ≤10%, and the accuracy of the method was 

considered acceptable when the inter-session calculated mean concentration at each 

QC level had a %RE value within ± 10%. 

The calculated concentrations and the associated intra- and inter-session statistics for the 

acetaldehyde assay QC samples are summarized in Table 2. The inter-session 

variability (RSD) of the calculated concentrations of each QC sample (precision) ranged 

from 5.7% to 7.9% RSD. The inter-session mean concentrations of the QC samples had 

%RE values (accuracy) ranging from -5.0% to -3.4%. Based on the stated criteria, the 

precision and accuracy of the acetaldehyde assay were acceptable. 

4.4. ASSAY RUGGEDNESS

Assay ruggedness, as required by WIL Research SOP, was successfully demonstrated for 

this method because at least 2 of the 3 validation sessions were performed by different 

analysts. 

4.5. ASSAY ACCEPTABILITY

In addition to the experimental samples, each analytical session consisted of (but was not 

limited to) calibration standards at 5 concentrations and triplicate QC samples prepared at 

each of 3 concentrations. In this study, the formulations were prepared at target 

acetaldehyde concentrations of 1 and 5 mg/mL, and the QC samples were prepared at 

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nominal acetaldehyde concentrations of 1.00, 3.00, and 5.00 mg/mL. For an analytical 

session to be considered valid, at least two-thirds of the calculated QC concentrations 

with at least 1 sample at each concentration had to be 90% to 110% of the nominal QC 

concentration. All reported results were from analytical sessions that met the acceptance 

criteria.

4.6. TEST SUBSTANCE STABILITY IN CALIBRATION STANDARDS

Calibration standards prepared at 10.0 and 50.0 μg/mL and analyzed on 

14 December 2011 were stored at room temperature for a minimum of 5 days 

before being re-analyzed to assess test substance stability. The mean post-storage 

concentrations were 90.8% and 99.5% of the pre-storage values (Table 3), which met the 

protocol-specified requirement for stability, i.e., the mean post-storage concentration was 

not <90% of the pre-storage value.

4.7. TEST SUBSTANCE STABILITY IN PROCESSED SAMPLES

QC samples prepared at nominal test substance concentrations of 1.00 and 5.00 mg/mL 

were processed and analyzed on 14 December 2011. The processed samples were stored 

at room temperature for a minimum of 5 days before being re-analyzed to assess test 

substance stability. The mean post-storage concentrations were 97.9% and 99.2% of the 

pre-storage values (Table 3), which met the previously stated protocol-specified 

requirement for stability.

4.8. TEST SUBSTANCE TIME-ZERO CONCENTRATION ASSESSMENT OF 

FORMULATIONS

Duplicate samples from the middle strata of formulations prepared on 19 December 2011 

at target concentrations of 1 and 5 mg acetaldehyde/mL were analyzed to verify test 

substance concentration acceptability in both glass containers and drinking water bottles. 

The results of the time-zero concentration acceptability assessments are presented in 

Table 4, with the mean concentrations and overall statistics summarized in the following 

table.

 Page 21 of 60
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

Time-Zero Concentration Assessment of the 19 December 2011 Formulations

Glass Containers Drinking Water Bottles

Low Group

(1 mg/mL)

High Group

(5 mg/mL)

Low Group

(1 mg/mL)

High Group

(5 mg/mL)

Mean Concentration (mg/mL) 0.921 4.78 0.934 5.20

SD 0.014 0.11 0.0098 0.11

RSD (%) 1.6 2.3 1.1 2.2

Mean Concentration % of Target 92.1 95.7 93.4 104

The analyzed formulations met the protocol-specified requirement for time-zero 

concentration acceptability for DI water formulations, i.e., the analyzed concentration 

was 90% to 110% of the target concentration.

4.9. TEST SUBSTANCE STABILITY OF FORMULATIONS IN GLASS 

CONTAINERS

The formulations prepared and analyzed on 19 December 2011 were stored in glass 

containers at room temperature for 24, 48, and 72 hours before being re-analyzed to 

assess test substance stability. The results of the stability analysis are presented in 

Table 5, Table 6, and Table 7. The mean concentrations and percent of time-zero are 

summarized in the following table. 

Mean Concentration, mg/mL (% of Time-Zero)

Storage Condition Storage Duration Low Group High Group

(1 mg/mL) (5 mg/mL)

Room 

Temperature

24 Hours 0.871 (94.5) 4.49 (93.9)

48 Hours 0.910 (98.8) 4.59 (95.9)

72 Hours 0.818 (88.8) 4.65 (97.2)

The post-storage test substance concentrations ranged from 93.9% to 98.8% of the 

pre-storage values, which met the previously stated protocol-specified requirement for 

stability with the following exception. The low group (1 mg/mL) formulation prepared 

 Page 22 of 60
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

on 19 December 2011 and stored at room temperature for 72 hours was 88.8% of the 

time-zero concentration which did not meet the stated criteria for stability.

4.10. TEST SUBSTANCE COMPATIBILITY OF FORMULATIONS IN DRINKING 

WATER BOTTLES

The formulations prepared and analyzed on 19 December 2011 were stored in drinking 

water bottles at room temperature for 24, 48, and 72 hours (in a position similar to that 

for dose administration) before being re-analyzed to assess test substance compatibility. 

The results of the compatibility analysis are presented in Table 8, Table 9, and Table 10. 

The mean concentrations and percent of post-storage concentration in glass containers are 

summarized in the following table. 

Mean Concentration, mg/mL (% of 

Post-Storage Concentration in Glass Containers)

Storage Condition Storage Duration Low Group High Group

(1 mg/mL) (5 mg/mL)

Room 

Temperature

24 Hours 0.878 (101) 4.54 (101)

48 Hours 0.899 (98.8) 4.61 (101)

72 Hours 0.925 (113) 4.96 (107)

The post-storage test substance concentrations ranged from 98.8% to 113% of the glass 

container post-storage values, which met the protocol-specified requirement for 

compatibility, i.e., the post-storage assay concentration was not <95% of the 

post-storage concentration of the formulation stored in glass containers.

 Page 23 of 60
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

5. CONCLUSIONS

A validated HPLC/UV method for the analysis of acetaldehyde concentration in DI water 

formulations and test substance ranging in concentration from 1.00 to 5.00 mg/mL was 

validated in this study. Assay specificity/selectivity, calibration reproducibility, 

precision, accuracy, ruggedness, and test substance stability in calibration standards and 

processed QC samples stored at room temperature for a minimum of 5 days were 

assessed and validated, satisfying protocol-specified criteria.

Formulations prepared at target test substance concentrations of 1 and 

5 mg acetaldehyde/mL met the protocol-specified requirement for concentration 

acceptability and, after 24, 48, and 72 hours of room temperature storage in glass 

containers, stability with the following exception. The low group (1 mg/mL) formulation 

prepared on 19 December 2011 and stored at room temperature for 72 hours was 88.8% 

of the time-zero concentration which did not meet the stated criteria for stability.

Formulations prepared at target test substance concentrations of 1.00 and 

5.00 mg acetaldehyde/mL met the protocol-specified requirement for formulation 

compatibility with drinking water bottles following 24, 48, and 72 hours of room 

temperature storage.

 Page 24 of 60
WIL-876002 Acetaldehyde 

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc. 

6. REPORT REVIEW AND APPROVAL 

Report Approved by: 

ManagerResearch Chemist, 

Analytical Chemistry 

Study Director 

Report Reviewed by: 

LA 

Eric S. Bodle, PhD 

Assistant Director, Analytical Chemishy 

Page 25 of 60 
WIL-876002 

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc. 

7. QUALITY ASSURANCE STATEMENT 

Date(s) of 

Insnection(s1 

03-Nov-2011 

20-Dec-2011 

20-Dec-2011 

23-Feb-2012, 

24-Feb-2012 

27-Feb-20 12, 

28-Feb-2012 

12-Mar-2012 

25-Feb-2013 

27-Feb-20 13 

Phase Insneckxi 

Protocol Review 

Protocol Amendment I Review 

Test Article Analysis 

Study Records (A-I) 

Analytical Chemistry Report 

Audited Analytical Chemistry Report 

Protocol Amendment 2 

Final Report 

Date(s) Findings 

Reported to 

Studv Direc tor 

03-Nov-2011 

20-Dec-2011 

20-Dec-2011 

24-Feb-2012 

28-Feb-2012 

12-Mar-2012 

25-Feb-2013 

27-Feb-20 13 

Acetaldehyde 

Date(s) 

Findings 

Reported to 

Manaeement 

20-Dec-201 I 

16-Jan-20 12 

16-Jan-20 12 

I I-Mar-2012 

I I-Mar-2012 

12-Mar-2012 

25-Feb-2013 

27-Feb-20 13 

This study was inspected in accordance with the applicable GLP Regulations, the WIL 

Research SOPS, and the protocol as approved by the Sponsor, with the following 

exception. The data located in Appendix B (Certificate of Analysis) were the 

responsibility of the Sponsor. A yearly internal facility inspection is conducted by the 

WIL Research Quality Assurance Department. 

This report accurately reflects the data generated during the study. The methods and 

procedures used in the study were those specified in the protocol and the WIL Research 

SOPS. 

o\ Mur.n:\3 

Date 

L 

Ehdeth B. Crookshank, BS 

Quality Assurance Representative 

Page 26 of 60 
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

8. DATA RETENTION

The raw data, the retention sample(s) if applicable, pertinent electronic storage media, 

and the original final report are retained in the WIL Research Archives in compliance 

with regulatory requirements.

 Page 27 of 60
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

9. ABBREVIATIONS

The following abbreviations may apply to this report:

μ - micro

μL - microliter

ACN - acetonitrile

btm - bottom

conc. - concentration

DI - deionized

ESI+ - positive electrospray ionization

FA - formic acid

FDA - Food and Drug Administration

g - gram

GLP - Good Laboratory Practices

HPLC - high performance liquid chromatography

hr - hour(s)

IS - internal standard

kg - kilogram

L - liter

LLOQ - lower limit of quantitation

MC - methylcellulose

mg - milligram

mL - milliliter

mm - millimeter

msec - milliseconds

MS - mass spectrometry

NA - not applicable

ND - not detected

ng - nanogram

nm - nanometer

OECD - Organisation for Economic Cooperation and Development

ppm - parts per million

QC - quality control

%RE - percent relative error

RSD - relative standard deviation

SD - standard deviation

SOP - standard operating procedure

UV - ultraviolet

v - volume

w - weight

WIL Research - WIL Research Laboratories, LLC

 Page 28 of 60
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

TABLES 1 - 10

 Page 29 of 60
Concentration (μg/mL) 10.0 20.0 25.0 40.0 50.0

Set 1 10.0 20.1 24.5 40.5 50.1

(14Dec2011) 10.0 19.9 25.8 40.5 49.7

Ruggedness 9.95 19.3 25.2 39.7 49.5

Mean 10.01 19.78 25.2 40.3 49.8

SD 0.049 0.41 0.66 0.51 0.31

%RSD 0.49 2.1 2.6 1.3 0.62

%RE 0.099 -1.1 0.60 0.63 -0.38

Set 2 10.0 19.95 25.1 40.7 48.8

(19Dec2011) 9.98 20.10 24.4 40.8 50.6

10.1 20.25 24.5 39.5 50.2

Mean 10.0 20.1 24.7 40.3 49.8

SD 0.077 0.15 0.36 0.72 0.93

%RSD 0.77 0.74 1.5 1.8 1.9

%RE 0.43 0.48 -1.3 0.82 -0.31

Set 3 10.2 20.4 25.4 39.5 *30.5

(20Dec2011) 10.0 20.2 25.3 38.3 50.3

10.0 20.0 24.1 40.8 51.0

Mean 10.0 20.2 24.9 39.5 50.6

SD 0.014 0.19 0.75 1.2 0.46

%RSD 0.14 0.96 3.0 3.1 0.91

%RE -0.1 0.9 -0.25 -1.2 1.3

Interset Statistics

n 99998

Mean 10.0 20.0 24.9 40.0 50.0

SD 0.055 0.30 0.57 0.85 0.67

%RSD 0.54 1.5 2.3 2.1 1.3

%RE 0.16 0.08 -0.30 0.083 0.057

*Sample deemed a statistical outlier and will not be used in statistical calcuations.

876002 results2.xls I

Printed: 12Mar2012 10:37 AM

Table 1. Back-Calculated Concentrations of the Validation Calibration Standards

WIL-876002 Acetaldehyde 

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc. 

 Page 30 of 60
Concentration (mg/mL) 1.00 3.00 5.00

Set 1 0.936 3.08 5.19

(14Dec2011) 1.04 3.12 5.24

Ruggedness 0.946 2.97 5.26

Mean 0.976 3.06 5.23

SD 0.060 0.077 0.036

%RSD 6.1 2.5 0.68

%RE -2.4 1.9 4.6

Set 2 0.985 2.75 4.65

(19Dec2011) 1.02 2.75 4.27

0.784 2.76 4.40

Mean 0.930 2.76 4.44

SD 0.13 0.0046 0.193

%RSD 14 0.17 4.3

%RE -7.0 -8.2 -11.2

Set 3 0.970 2.77 4.60

(20-21 Dec 2011) 0.999 2.74 4.69

1.01 2.70 4.70

Mean 0.991 2.73 4.66

SD 0.019 0.035 0.051

%RSD 1.9 1.3 1.1

%RE -0.87 -8.9 -6.8

Interset Statistics

n 999

Mean 0.966 2.85 4.78

SD 0.076 0.16 0.37

%RSD 7.9 5.7 7.7

%RE -3.4 -5.0 -4.5

876002 results2.xls II

Printed: 12Mar2012 10:37 AM

Table 2. Calculated Concentrations of the Validation Quality Control Samples

WIL-876002 Acetaldehyde 

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc. 

 Page 31 of 60
Theo.

Time Point Conc Ref # Run # Conc

(μg/mL) ( 876002 - ) (μg/mL)

Calibration Standards

t=0 10.0 37 - 1 9 10.0

t=6 37 - 1 223 9.05

t=0 10.0 37 - 2 10 10.0

t=6 37 - 2 224 9.17

t=0 50.0 37 - 14 22 49.7

t=6 37 - 14 225 49.2

t=0 50.0 37 - 15 23 49.5

t=6 37 - 15 226 49.5

Theo.

Time Point Conc Ref # Run # Conc

( mg/mL ) ( 876002 - ) ( mg/mL )

QC Samples

t=0 1.00 40 - 2 27 0.936

t=6 40 - 2 228 0.909

t=0 1.00 40 - 3 28 1.04

t=6 40 - 3 229 1.03

t=0 5.00 40 - 9 34 5.24

t=6 40 - 9 230 5.22

t=0 5.00 40 - 10 35 5.26

t=6 40 - 10 231 5.20

N/A = Not applicable

876002 results2.xls pss1d(rt)

Printed: 03/12/12 10:42 AM

Percent of

Overall

99.5

90.8

(%)

Time Zero

N/A

99

97

N/A

99

N/A 99.2

99

Time Zero Time Zero

(%) (%)

N/A 97.9

N/A

100

Percent of

90.1

N/A

91.4

N/A

99.0

Time Zero

(%)

N/A

Table 3. Minimum 5-Day Room Temperature Stability Analysis of the

14 December 2011 Calibration Standards and Processed Quality Control Samples

Overall

Percent of Percent of

WIL-876002 Acetaldehyde 

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc. 

 Page 32 of 60
Dose Analyzed Percent Mean Mean Conc

Group Strata Conc Ref # Run # Conc of Target Conc SD RSD % of Target

( mg/mL ) ( mg/mL ) (%) ( mg/mL ) (%) (%)

Glass Container Formulations

Low Mid 1 58 - 1 156 0.911 91.1 0.921 0.014 1.6 92.1

58 - 2 157 0.931 93.1

High Mid 5 58 - 3 158 4.71 94.1 4.78 0.11 2.3 95.7

58 - 4 159 4.86 97.2

Drinking Bottle Formulations

Low Mid 1 61 - 1 162 0.941 94.1 0.934 0.0098 1.1 93.4

61 - 2 163 0.927 92.7

High Mid 5 61 - 3 164 5.12 102 5.20 0.11 2.2 104

61 - 4 165 5.28 106

876002 results2.xls 1C 

Printed: 03/12/12 10:37 AM

Table 4. Concentration Assessment of the 19 December 2011 Formulations

(Analyzed 19 December 2011)

( 876002 - )

Acetaldehyde Working Group of the WIL-876002 Acetaldehyde 

Vinyl Acetate Council, Inc. 

Page 33 of 60
Dose Analyzed Percent Mean Mean Conc Percent of

Group Strata Conc Ref # Run # Conc of Target Conc SD RSD % of Target Time Zero

( mg/mL ) ( mg/mL ) (%) ( mg/mL ) (%) (%)

Glass Container Formulations

Low Mid 1 74 - 1 218 0.844 84.4 0.871 0.038 4.4 87.1 94.5

74 - 2 219 0.898 89.8

High Mid 5 74 - 3 220 4.44 88.8 4.49 0.075 1.7 89.8 93.9

74 - 4 221 4.54 90.9

Group

Low

High

876002 results2.xls 2 24hr RT STB glass jars

Printed: 03/12/12 10:37 AM

Table 5. 24-Hour Room Temperature Stability Assessment of the 19 December 2011 Formulations (Glass Container)

(Analyzed 20 December 2011)

( 876002 - )

Time Zero Conc

(mg/mL)

0.921

4.78

Acetaldehyde Working Group of the WIL-876002 Acetaldehyde 

Vinyl Acetate Council, Inc. 

Page 34 of 60
Dose Analyzed Percent Mean Mean Conc Percent of

Group Strata Conc Ref # Run # Conc of Target Conc SD RSD % of Target Time Zero

( mg/mL ) ( mg/mL ) (%) ( mg/mL ) (%) (%)

Glass Container Formulations

Low Mid 1 84 - 1 336 0.917 91.7 0.910 0.010 1.1 91.0 98.8

84 - 2 337 0.903 90.3

High Mid 5 84 - 3 338 4.55 91.1 4.59 0.046 1.0 91.7 95.9

84 - 4 339 4.62 92.4

Group

Low

High

876002 results2.xls 3 48hr RT STB glass jars 

Printed: 03/12/12 10:37 AM

Table 6. 48-Hour Room Temperature Stability Assessment of the 19 December 2011 Formulations (Glass Container)

(Analyzed 21 December 2011)

( 876002 - )

Time Zero Conc

(mg/mL)

0.921

4.78

Acetaldehyde Working Group of the WIL-876002 Acetaldehyde 

Vinyl Acetate Council, Inc. 

Page 35 of 60
Dose Analyzed Percent Mean Mean Conc Percent of

Group Strata Conc Ref # Run # Conc of Target Conc SD RSD % of Target Time Zero

( mg/mL ) ( mg/mL ) (%) ( mg/mL ) (%) (%)

Glass Container Formulations

Low Mid 1 98 - 1 348 0.709 70.9 0.818 0.15 19 81.8 88.8

98 - 2 349 0.926 92.6

High Mid 5 98 - 3 350 4.42 88.5 4.65 0.32 6.9 93.0 97.2

98 - 4 351 4.88 97.5

Group

Low

High

876002 results2.xls 4 72hr RT STB glass jars 

Printed: 03/12/12 10:37 AM

4.78

Table 7. 72-Hour Room Temperature Stability Assessment of the 19 December 2011 Formulations (Glass Container)

(Analyzed 22 December 2011)

( 876002 - )

Time Zero Conc

(mg/mL)

0.921

Acetaldehyde Working Group of the WIL-876002 Acetaldehyde 

Vinyl Acetate Council, Inc. 

Page 36 of 60
Dose Analyzed Percent Mean Mean Conc Percent of Percent of Glass

Group Strata Conc Ref # Run # Conc of Target Conc SD RSD % of Target Time Zero Post-Storage

( mg/mL ) ( mg/mL ) (%) ( mg/mL ) (%) (%)

Drinking Bottle Formulations

Low Mid 1 71 - 1 213 0.881 88.1 0.878 0.0043 0.49 87.8 94.0 101

71 - 2 214 0.875 87.5

High Mid 5 71 - 3 215 4.51 90 4.54 0.049 1.1 90.9 87.4 101

71 - 4 216 4.58 92

Group

Low

High

876002 results2.xls 2 24hr RT STB drinking bottles

Printed: 03/12/12 10:37 AM

Table 8. 24-Hour Room Temperature Stability Assessment of the 19 December 2011 Formulations (Drinking Bottles)

(Analyzed 20 December 2011)

( 876002 - )

Time Zero Conc

(mg/mL)

0.934

Glass Post-Storage Conc

(mg/mL)

0.871

5.20 4.49

Acetaldehyde Working Group of the WIL-876002 Acetaldehyde 

Vinyl Acetate Council, Inc. 

Page 37 of 60
Dose Analyzed Percent Mean Mean Conc Percent of Percent of Glass

Group Strata Conc Ref # Run # Conc of Target Conc SD RSD % of Target Time Zero Post-Storage

( mg/mL ) ( mg/mL ) (%) ( mg/mL ) (%) (%)

Drinking Bottle Formulations

Low Mid 1 81 - 1 330 0.920 92.0 0.899 0.030 3.3 89.9 96.3 98.8

81 - 2 331 0.878 87.8

High Mid 5 81 - 3 332 4.55 91 4.61 0.088 1.9 92.3 88.8 101

81 - 4 333 4.68 94

Group

Low

High

876002 results2.xls 3 48hr RT STB drinking bottle

Printed: 03/12/12 10:37 AM

Table 9. 48-Hour Room Temperature Stability Assessment of the 19 December 2011 Formulations (Drinking Bottles)

(Analyzed 21 December 2011)

( 876002 - )

Time Zero Conc Glass Post-Storage Conc

(mg/mL) (mg/mL)

0.934 0.910

5.20 4.59

Acetaldehyde Working Group of the WIL-876002 Acetaldehyde 

Vinyl Acetate Council, Inc. 

Page 38 of 60
Dose Analyzed Percent Mean Mean Conc Percent of Percent of Glass

Group Strata Conc Ref # Run # Conc of Target Conc SD RSD % of Target Time Zero Post-Storage

( mg/mL ) ( mg/mL ) (%) ( mg/mL ) (%) (%)

Drinking Bottle Formulations

Low Mid 1 95 - 1 342 0.910 91.0 0.925 0.022 2.4 92.5 99.0 113

95 - 2 343 0.940 94.0

High Mid 5 95 - 3 344 5.01 100 4.96 0.083 1.7 99.1 95.3 107

95 - 4 345 4.90 98

Group

Low

High

876002 results2.xls 4 72hr RT STB bottle

Printed: 03/12/12 10:37 AM

0.934 0.818

5.20 4.65

Table 10. 72-Hour Room Temperature Stability Assessment of the 19 December 2011 Formulations (Drinking Bottles)

(Analyzed 22 December 2011)

( 876002 - )

Time Zero Conc Glass Post-Storage Conc

(mg/mL) (mg/mL)

Acetaldehyde Working Group of the WIL-876002 Acetaldehyde 

Vinyl Acetate Council, Inc. 

Page 39 of 60
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

APPENDIX A

Study Protocol and Deviations

 Page 40 of 60
WIL-876002 Acetaldehyde

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.

DEVIATIONS FROM THE PROTOCOL

This study was conducted in accordance with the protocol and protocol amendments, 

except for the following.

Protocol Section 4.1.9 states that a reserve sample will be taken; however, a

sample was not taken.

Reason for Deviation: Due to the high volatility of the test substance, no reserve 

sample was collected for storage.

Protocol Section 6.3.5 states that formulations that remained after sampling will 

be stored in bulk glass containers at room temperature and refrigerated conditions. 

Formulations that were to be stored refrigerated in bulk glass containers processed 

for 24-, 48-, and 72-hour stability were never performed. 

Reason for Deviation: Per Study Director, these assessments were not needed 

for this study.

These deviations did not negatively impact the quality or integrity of the data nor the 

outcome of the study.

3DJHRI
 Page 42 of 60
Page 2 of 2 

Approval: 

Sponsor% approval was obtained via email on 22 February 2013. 

WIL Research 

WE476002 

Protocol Amendment 2 

ate 

Study Director 

--_- Assistant Director, Analytical Chemistry 

Acetaldehyde Working Group 

 Page 43 of 60
 Page 44 of 60
 Page 45 of 60
 Page 46 of 60
~ 

DepartmentaVStudy Director Notification 

Study Director: Robert Boes 

Study NO.: WIL-876002 

Please note the following: 

Protocol sections 6.3.3, 6.3.5, and 6.3.6: 

The concentrations on study will be changed from I%, 3% and 5% to 1 mg/mL, 3 

mg/mL, and 5 mglmL. 

Reason for change: 

To correct the wording in the protocol to match the actual concentrations 

SDonsor approval on: I 3 Orc 2011 via em;r-t or NA 

mProtocol amendment forthcoming 

0 Clarification of procedure, no protocol amendment required 

Study Director: Date: 13 OCc 20ll 

Original: QAU 

Copies as per protocol distribution 

A-I 86-9 

 Page 47 of 60
.a. ...I. 

1.1, 

Page 1 of 11 

WIL-876002 

November 7,20 11 

PROTOCOL 

AN ANALYTICAL METHOD VALIDATION, COMPATIBILITY, 

AND STABILITY STUDY OF ACETALDEHYDE IN DEIONIZED WATER 

Sponsor: 

Acetaldehyde Working Group 

of the Vinyl Acetate Council, Inc 

1250 Connecticut Ave, NW 

Suite 700 

Washington, DC, 20036 

Testing Facility: 

WIL Research Laboratories, LLC 

1407 George Road 

Ashland, OH 44805-8946 

 Page 48 of 60
WIL-876002 

Page 2 of 11 November 7,201 1 

1 OBJECTIVE: 

To develop and validate a method for the determination of acetaldehyde 

concentration in deionized (DI) water using high performance liquid chromatography 

(HPLC). DI water formulations prepared at the lowest and highest test substance 

concentrations anticipated for future studies will be assessed for test substance 

compatibility in various water bottles. Test substance stability in DI water after 24 to 

32 hours of room temperature or refrigerated storage will also be assessed. 

This study will be conducted in compliance with the U.S. Food and Drug 

Administration (FDA) Good Laboratory Practice Standards, 21 CFR Part 58 (October 

5, 1987), the U.S. EPAITSCA, 40 CFR Part 792, and the OECD, [C(97)186/Final], 

Good Laboratory Practice Standards. The study will also be conducted in accordance 

with the protocol and WIL Research Standard Operating Procedures. 

2 PERSOmL INVOLVED IN THE STUDY: 

2.1 Sponsor Re~resentative: 

Robert Fensterheim, MPH 

Acetaldehyde Working Group 

1250 Connecticut Ave., NW 

Suite 700 

Washington, DC 20036 

Tel: (202) 419-1500 

E-mail: rfensterheim@regnet.com 

2.2 Study Monitor: 

Willem Faber, PhD, DABT 

WFTC, LLC 

1338 Strong Rd., Victor, NY 14564 

Tel: (585) 742-1568 

E-mail: wfaber@msn.com 

2.3 WIL Study Director: 

Robert E. Boes, Jr., MS 

Associate Research Chemist, Analytical Chemistry 

Tel: (419) 289-8700 

Fax: (419) 289-3650 

E-mail: robert.boes@wilresearch.com 

 Page 49 of 60
WIL-876002 

Page 3 of 11 November 7,201 1 

2.4 WIL Departmental Responsibilities: 

Eric S. Bodle, PhD 

Assistant Director, Analytical Chemistry 

Emergency Contact 

Tel: (419) 289-8700 

Fax: (419) 289-3650 

E-mail: ebodle@wilresearch.com 

Steven D. Barkyournb, DVM, PhD, DACVP 

Senior Vice-President, Global Operations - Safety Assessment 

Michael D. Schlosser, PhD, DABT 

Vice President, Analytical, Metabolism, 

and In Vitro Toxicology Services 

Heather L. Johnson, BS RQAP-GLP 

Manager, Quality Assurance 

Robert A. Wally, BS 

Operations Manager, Reporting and 

Technical Support Services 

3 STUDY SCHEDULE: 

Proposed Experimental Start Date: November 20 1 1 

Proposed Experimental Termination Date: December 20 1 1 

Proposed Audited Report Date: Typically 6 weeks after the 

completion of validation activities. 

4 TEST SUBSTANCE INFORMATION: 

The supplier is responsible for providing documentation of test substance 

characterization, typically a certificate of analysis or testing. 

4.1 Test Substances: 

4.11 Identifications: 

Acetaldehyde 

 Page 50 of 60
WIL-876002 

Page4of 11 November 7,201 1 

4.1.2 Supplier 

Sigma Aldrich 

4.1.3 Lot Numbers: 

4.1.4 Purity: 

4.1.5 Stability: 

The test substance is considered stable under the storage conditions 

provided by the Supplier. 

4.1.6 Physical Description: 

To be documented by WIL Research Laboratories, LLC. 

4.1.7 Storage Conditions: 

Refrigerated, protected from light, under nitrogen cap 

4.1.8 Expiration Date: 

October 4,2012 

4.1.9 Reserve Samples: 

Reserve samples of the test substance will be taken in accordance with 

WIL Standard Operating Procedures and stored in the Archives at WIL 

Research Laboratories, LLC indefinitely, unless otherwise specified. 

4.1.10 Personnel Safety Information: 

A Material Safety Data Sheet (MSDS) of the compound has been 

provided by Supplier, and appropriate safety precautions will be 

followed based on the MSDS. 

5 TEST SYSTEM: 

Deionized @I) water 

 Page 51 of 60
WIL-876002 

Page 5 of 11 November 7,201 1 

6 EXPERIMENTAL DESIGN: 

6.1 Overview of the Study: 

Acetaldehyde is the test substance for this study and will be referred to as the 

analyte. The method to be validated is for the determination of analyte 

concentration in DI water formulations. This study will provide the necessary 

data that demonstrate the analytical methods as valid. 

6.2 Method Details 

6.2.1 Instrument 

A HPLC equipped with a variable wavelength or diode array detector, 

an autosampler, and Dionex Chromeleon Software, or equivalent 

system. Possible systems include: 

Agilent 1 100 HPLC System 

Dionex UltiMate 3000 HPLC System 

6.2.2 Matrix: 

DI water 

6.2.3 Method: 

The method validation activities include two phases: (1) method 

evaluation and development, and (2) formal method validation. 

Method evaluation of sponsor-supplied methodology usually includes 

(but is not limited to) the following activities: (1) the analysis of 

standards prepared in an appropriate solvent to establish 

chromatography, including retention times, resolution, sensitivity, and to 

check proportionality of response; (2) the analysis of the analyte 

prepared in the matrix to confirm the presence or absence of 

interferences, to evaluate potential stability limitations, and to evaluate 

response proportionality. Client supplied methodology and other 

literature will be used as a starting point for method 

evaluation/development. Method development/evaluation will not be 

audited. 

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WIL-876002 

Page 6 of 11 November 7,201 1 

6.3 Studv Details and Criteria: 

6.3.1 Specificity: 

The specificity of the methods will be determined by analyzing 

representative blank samples. The retention time window(s) 

corresponding to the analyte will be examined for interferences and, if 

needed, appropriate efforts to minimize interfering peaks will be taken 

such as: adjustment or change of chromatographic parameters to 

maximize resolution of interference and analyte peaks; use of a more 

analyte-specific wavelength; and change in sample preparation 

procedure to minimize the presence of the interference in the sample to 

be analyzed. The success of these efforts will be determined when the 

method validation either passes or fails the accuracy and precision 

acceptance criteria for calibration and quality control samples. 

6.3.2 Calibration Reproducibility: 

A minimum of 3 validation sessions (for each test substance) will be 

performed to validate the method for the determination of analyte 

concentration in DI water formulations. For each validation session, at 

least triplicate calibration samples at a minimum of 5 different analyte 

concentrations will be prepared and analyzed. The concentration of the 

calibration samples and the regression model used for the regression 

analysis will be specified in the written method to be validated. The 

results of the regression analysis will be used to back-calculate the 

calibration sample concentrations. The inter-session back-calculated 

concentration data at each calibration level must be precise (relative 

standard deviation [RSD] less than or equal to lo%, except at the lowest 

concentration level where it should not exceed 15%), and accurate 

(relative error [RE] is within * 10% except at the lowest concentration 

level where it should not exceed * 15%). 

6.3.3 Accuracy and Precision: 

Quality control samples will be prepared at a minimum of 

3 concentrations (for each test substance) in blank matrix - one near the 

lowest, one near the middle and one near the highest formulation 

concentration expected for future studies (I%, 3% and 5%). The 

concentration of the QC samples will be specified in the written method 

to be validated. At least 3 replicate quality control samples at each 

concentration level will be analyzed with the calibration standards 

during each validation session. The inter-session accuracy and precision 

will be established based on the analyzed concentrations of the quality 

control samples. The inter-session analyzed concentration data at each 

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WIL-876002 

Page 7 of 11 November 7,201 1 

QC level must be precise (RSD less than or equal to lo%), and accurate 

(RE is within k 10%). 

6.3.4 Stability: 

The room temperature (or autosampler temperature if a cooled 

autosampler proves appropriate and necessary for adequate analyte 

stability) stability of processed samples will be evaluated over a 

minimum of 24 hours. 

If a significant degradation (>lo% reduction in the mean analyte 

concentration or response fiom the time zero samples) occurs under the 

tested conditions, then special precautions should be taken. 

6.3.5 Test Substance Stability in DI Water Formulations: 

Stability of test substance in DI water formulations will be determined. 

Formulations at test substance concentrations representing the lowest 

and highest anticipated concentrations (1% and 5%) will be prepared. 

For the time-zero concentration assessment, samples (in at least 

duplicate) will be collected from the formulations on the day of 

preparation and analyzed to assess test article concentration 

acceptability. In order for the test substance formulations to be 

considered acceptable, the mean concentration must be within 90% to 

1 10% of the target concentration. 

Formulations that remain after sampling will be stored in bulk glass 

containers at room temperature and reficigerated conditions. Following 

storage, samples (in at least duplicate) will be collected fiom the mid 

strata of each preparation and analyzed. Analysis will be conducted 

following storage for 24 hours, if stable, subsequent analysis will be 

conducted after 48 hours of storage; and then if still stable, again at 

72 hours. Two samples fiom each concentration will be analyzed to 

assess test substance stability. In order for the test substance to be 

considered stable, the post-storage assay concentration cannot be less 

than 90% of the pre-storage concentration. All back-up samples 

collected will be stored at room temperature and may be discarded after 

the Study Director's approval of the analytical results. 

6.3.6 Test Substance Formulation Compatibility with Drinking Bottles: 

Formulations that remain after sampling for the concentration 

assessments (1% and 5%) will be stored in various drinking water 

bottles in a position similar to that for dose administration. The 

formulations will remain in the bottles for 24, 48 and 72 hours at room 

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WIL-876002 

Page 8 of 11 November 7,201 1 

temperature conditions. Following storage, samples (in at least 

duplicate) will be collected fiom the mid strata of each bottle. Two 

samples fiom each concentration will be analyzed to assess test 

substance formulation compatibility. In order for the test substance 

formulations to be considered compatible with the bottles, the poststorage assay concentration cannot be less than 95% of the post-storage 

concentration of the formulation stored in glass containers. All back-up 

samples collected will be stored at room temperature and may be 

discarded after the Study Director's approval of the analytical results. 

7 QUALITY ASSURANCE: 

The study will be audited by the WIL Quality Assurance Unit while in progress to 

assure compliance with Good Laboratory Practice regulations, adherence to the 

protocol and to WIL Standard Operating Procedures. The raw data and draft report 

will be audited by the WIL Quality Assurance Unit prior to submission to the Sponsor 

to assure that the final report accurately describes the conduct and the findings of the 

study. 

This study will be included on the WIL master list of regulated studies. 

8 RECORDS TO BE MAINTAINED: 

All original raw data records, as defined by WIL SOPs and the applicable GLPs, will 

be stored in the Archives at WIL Research Laboratories, LLC. Records to be retained 

will include, but are not limited to the following: 

Protocol and protocol amendments 

The original chromatograms, spectra and other instrument generated data 

Calculations of concentration levels and appropriate test parameters 

9 WORK PRODUCT: 

The Sponsor will have title to all documentation records, raw data, and other work 

product generated during the performance of the study. All work product, including 

raw paper data and pertinent electronic storage media, will be retained at no charge 

for a period of six months following issuance of the final report in the Archives at 

WIL Research Laboratories, LLC. Thereafter, WIL Research Laboratories, LLC will 

charge a monthly archiving fee for retention of all work product. All work product 

will be stored in compliance with regulatory requirements. 

Any work product, including documents, and samples, that are required by this 

protocol, its amendments, or other written instructions of the Sponsor, to be shipped 

by WIL Research Laboratories, LLC to another location will be appropriately 

packaged and labeled as defined by WIL's SOPs and delivered to a common carrier 

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Page 9 of 11 November 7,201 1 

for shipment. WIL Research Laboratories, LLC will not be responsible for shipment 

following delivery to the common carrier. 

10 REPORTS: 

The final report will contain a summary, test substance data, methods and procedures, 

and an interpretation and discussion of the study results. The report will contain all 

information necessary to conform with current FDA, EPA, and OECD specifications. 

The contents of the report will be as follows: 

The study will be summarized in a formal report. 

Details of all experimental procedures will be described. Methods of calculation 

will be described. 

Sample preparation, chromatographic or other test conditions, calibration 

reproducibility, accuracy and precision will be detailed. 

Copies of chromatograms obtained in the analysis will be entered as appropriate. 

Any protocol or GLP deviations that may occur during the study. 

A compliance statement and a Quality Assurance Unit statement will be included. 

WIL Research Laboratories, LLC will provide one (1) electronic copy of an audited 

draft report, submitted in a timely manner upon completion of the validation activities 

prior to issuance of the final report. One (1) revision will be permitted as part of the 

cost of the study, from which Sponsor's reasonable revisions and suggestions will be 

incorporated into the Final Report as appropriate. Additional changes or revisions 

may be made, at extra cost. It is expected that the Sponsor will review the draft 

report and provide comments to WIL within a two (2) month time fiame following 

submission. WIL will submit the final report within one (1) month following receipt 

of comments. If the Sponsor's comments andlor authorization to finalize the report 

have not been received at WIL within one year following submission of the draft 

report, WIL may elect to finalize the report following appropriate written notification 

to the Sponsor. Two (2) copies of the final report [one paper copy (unbound) and one 

electronic copy (CDR)], will be provided; requests for additional copies of the final 

report may result in additional charges. 

11 PROTOCOL MODIFICATION: 

Modification of the protocol may be accomplished during the course of this study. 

However, no changes will be made in the study design without the verbal or written 

permission of the Sponsor. In the event that the Sponsor verbally requests or 

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WIL-876002 

Page 10 of 11 November 7,201 1 

approves a change in the protocol, such changes will be made by appropriate 

documentation in the form of a protocol amendment. All alterations of the protocol 

and reasons for the modification(s) will be signed by the Study Director and the 

Sponsor Representative. 

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12 PROTOCOLAPPROVAL: 

Sponsor approval received via em; f on 2 NOV 20ll . 

Acetaldehyde Working Group 

y lvo~ao~l 

~dert Fensterheim, MPH Date 

Sponsor Representative 

WE Research Laboratories, LLC 

7 Nsv 2011 

Date 

Study Director 

9/vod Zo\\ - EricS.Bodle,PhD Date 

Assistant Director, hal&xd Chemistry 

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WIL-876002 Acetaldehyde 

Acetaldehyde Working Group of the Vinyl Acetate Council, Inc. 

APPENDIX B 

Certificate of Analysis

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