**FINAL REPORT**

Study Title : An Anal yt ical Method Validation, Compatibility , and Stability

Study of Acetaldehy de in Deionized Water Laboratory

Project ID : WI L

- 876002 Author : Scott D. Freer, BA Test Guideline : Not Applicable Study Compl etion Date : 01 March 2013 Performing Anal yt ical Laboratory : WI L Research 1407 George Road Ashland, OH 44805

- 8946 Sponsor : Acetaldeh yd e Working Group of the Viny l Acetate Council, Inc

. 1250 Connecticut Ave, NW Suite 700 Washington, DC 20036 To tal Number of Pages : 60

**Page 1 of 60**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: Page 2 of 60WI L-876002 Acetaldeh yd e

Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### T **ABLE O F**  

### C **ONTENTS** 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

. WI L 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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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. Page 3.16

. WI L Res earch Computer Sy stems
................................ ............................. 15 3.16.1

. Reporting and Ancillary Sy stems
................................ .............................. 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**

**Page 4 of 60**WI L-876002 Acetaldeh yd e

Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### I **NDEX OF** 

### F **IGURES** Page 1. Representative Chromatogram of a 10.0 μg Acetaldeh yde/mL Calibration Standard

................................ ....... 16 2. Representative Chromatogram of a Processed 5.0 0 mg Acetaldeh yd e/mL Quality Control Sample

................................ .. 17 3. Representative Chromatogram of a Processed 5.00 mg Acetaldeh yd e/mL Formulation Sample................................

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

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

................................ ......... 18



### I **NDEX OF** 

### T **AB LES**  1

. Back

- Calculated Concentrations of the Validation Calibration Standards

................................ ................................ ................................ .... 30 2

. Calculated Concentrations of the Validation Quality Control Samples................................

................................ ................................ ...... 3 1 3

. Minimum 5

- Day

Room Temperature Stability Analy sis of the 14 December 2011 Calibration Standards and Processed Quality Control Samples

................................ ................................ ......................... 3 2 4

. Concentration Assessment of the 19 December 2011 Formulations

......... 3 3 5 . 24 -Hour Room Te mperature Stability Assessment of the 19 December 2011 Formulations (Glass Container)

................................ . 3 4 6 . 48

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

................................ . 3 5 7 . 72

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

................................ . 3 6 8 . 24

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

................................ 3 7 9 . 48

- Hour Room Temperature Stability Assessment of the 19 December 2011 Formulations (Drink ing Bottles)

................................ 38 10 . 72

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

................................ 39
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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### I **NDEX O F**  

### A **PPENDICES** Page A

. Study

Protocol and Deviat i ons ................................ ................................ ... 40 B

. Certificate of Anal ys is

................................ ................................ ............... 5 9

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 1. **S UMMAR** 

**Y** A high performance liquid chromatograph y method using ultraviolet absorbance detection at a wavelength of 360 nm for the determination of acetaldeh yd e concentration in deionized (DI) water formulations containing test substance ranging in concentration from 1 .00 to 5 .00 mg/mLw as validated in this study

. A lso in this study , test substance stability w as assessed in calibration standard s and processed quality control (QC) samples stored at room temperature for a minimum of 5 day s. In addition, test substance concentration acc eptability and, following 24 , 48 , and 72 hours of room temperature storage in glass containers , stability w ere assessed in DI water formulations prepared at target concentrations of 1 and 5 mga cetaldehy de/mL

. In addition, test substance formulation compatibility w ith drinking water bottles following 24, 48, and 72 hours of room temperature storage was assessed in DIw ater formulations prepared at target concentrations of 1 .00 and 5 .00 mg acetaldeh yd e/mL

. The acetaldeh yd e assay

p rocedure was validated in this study w ith 3 validation sessions. Quantitation was performed using calibration standards ranging in test substance concentration from 10.0 to 50.0 μg/mL

. T he mean back

- calculated standard concentrations had inter

- session variability r anging from 0. 5 4 % to 2.3% relative standard deviation (RSD) and percent relative error (%RE) ranging from

- 0. 30 % t o 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). A ssay p recision and accuracy

w ere verified by

the analy sis of QC samples prepared at 1.00, 3.00, and 5.00 mga cetaldeh yde/mL. T he 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 %. T he results met the protocol

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

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. The test substance in calibration standards and pr ocessed QC samples stored at room temperature for a min imum of 5 day s 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 a ssessment in DIw ater formulations prepared at target concentrations of 1 and 5 mg a cetaldeh yde/mLm et the protocol

- specified acceptance criteria for concentration acceptability , i.e. , the mean concentration was 90% to 110% of the target con centration. Assessment of test substance stability

i n glass containers following 24 , 48, and 72 hours of room temperature storage in DIw ater formulations prepared at target concentrations of 1 and 5 mg acetaldeh yde/mL met the previously s tated protocol

- specified requirement for stability w ith 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 stabilit y. The results of the test substance compatibility assessment with drinking water bottles following 24, 48, and 72 hours of room temperature storage in DIw ater formulations prepared at target concentrations of 1 and 5 mga cetaldeh yde/mLm et th e protocol

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

- storage assay c oncentration was not < 95% of the post
- storage concentration of the formulation stored in glass containers. 

### 2. **I NTRODUCTION**  This report provides a detailed description and validation of a high performance liquid chromatograph y (HPLC) method using ultraviolet (UV) absorbance detection at a wav elength of 360 nm for the determination of acetaldeh yd e concentration in deionized ( DI ) water formulations and test substance rangi ng in concentration from 1 .00 to 5 .00 mg/mL

. Assay s pecificit y/ selectivity, calibration reproducibility , precision, accuracy , ruggedness, and test substance stability in calibration standards and processed

**Page 8 of 60**WI L

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. QC samples stored at room temperatur e for a minimum of 5 day s were established and verified. Also in this study , DIw ater formulations prepared at target concentrations of 1 to 5 mg acetaldeh yd e/mLw ere anal yz ed to assess test substance concentration acceptability and, following 24, 48, and 72 hours of room temperature storage in glass containers, stability

. In addition, DIw ater formulations prepared at target concentrations of 1 to 5 mg acetaldeh yde/mL were analyzed to assess test substance compatibility with drinking water bottles follo wing 24, 48, and 72 hours of room temperature storage. The study protocol is presented in Appendix A

. A list of abbreviations potentially u sed in this report is presented in Section 9

. (Abbreviations)

. 

### 2.1. **K EY**  

### S **TUDY** 

### D **ATES Date(s)**  **Event(s)** 07 November 2011

................................ . Study Initiation Date 14 December 2011

................................ .. Experimental start ( starting ) date (first date of analysis) 2 3 December 2011

................................ .. Experimental termination (completion) date (last date of analy sis) 

### 2.2. **WIL R** **ESEARCH** 

### K**EY** 

### S **TUDY** 

### P **ERSONNEL** Robert E. Boes Jr. , MS Associate Research Chemist, Analy tical Chemistry Dennis J . Hoobler, BS Training Coordinator , Analy tical Chemistry Phong X. Pham, BS Chemist I I , Analy tical Chemistry Gregory A. Hawks, AS Group Supervisor , Reporting & Technical Support Services Melissa A. Hull, BS WIL Research

- US Manager, Computerized Sy stems Validation

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 3. **E XPERIMENT** 

**AL** 

### P **ROCEDURES** 

### - **M AT**  **ERIALS AND**  

### M **E T**  **H ODS**  

### 3.1. **T EST**  

### S **UBSTANCE AND**  

### V **EHICLE** 

### 3.1.1. **T EST**  

### S **UBSTANCE** 

### I **DENTIFICATION** The test substance , acetal dehy de, was received from Sigma

- Aldrich, Milwaukee, WIo n 0 4 October 2011 as follows:

Id entification Quantity Received Phy sical Description Acetaldeh yd e Lot no. SHBB4508V [WIL ID no. 110159] 20 Containers

Gross volume: 20 L Colorless liquid
Documentation regarding the purit y and stability of the test substanc e is on file with the Sponsor and WIL Research. AC ertificate of Analy sis for the test substance was provided by

the vendor and is presented in Appendix B

. T he purity o f the test substance was ≥99.5%. T he test substance was stored refrigerated, protected from light, under nitrogen cap and was considered stable under th ese condition s . 

### 3.1.2. **V EHICLE**  

### I **DENTIFICATION** The vehicle used in preparation of the test substance formulations was DI water. 

### 3.2. **D R** **INKING** 

### W **ATER** 

### B **OTTLES** The drinking water bottles used to determine compatibility w ith the DIw ater formulations were received 30 November 2011 as follows:

● 6 empty

4 00

- mLp ol yc arbonate bottles with silicone rings (list no. ACBT0402, received from Tecniplast USA Inc., West Chester, P A )
● 6 S/S caps with 2 balls (list no. ACCPS50, re ceived from Tecniplast USA Inc. )

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 3.3. **H IGH**  

### P **ERFORMANCE** 

### L **IQUID** 

### C **HROMATOGRAPHY** Instrument: Agilent 1100 liquid chromatograph equipped with a variable wavelength detector, autosampler , and Dionex Chromeleon ® software, or equivalent s ys tem Column: Zorb ax ODS column, 4.6 mm × 250 mm ., 5

- μm p article

- size Mobile Phase A: 6 0:40 (v/v) Acetonitrile (ACN) :DI w ater Flow Rate: 1.50 mL/minute Column Temperature: 25.0 ° C Detector: U V at 360 nm Injection Volume: 20.0 μL Retention Time: Approximately 5.2 minutes for a cetaldeh yd e Run Time: 15 minutes 

### 3.4. **P REPARATION OF**  

### M **OBILE** 

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

### 3.5. **P REPARATION OF**  

### 0 **.5 M**  **C ITRATE**  

### A **CID** A 0.5 M citrate acid solution was prepared by d issolving 96.05 g of citric acid in 1000 mLof DI water. 

### 3.6. **P REPARATION OF**  

### 0.5 **M S**  **ODIUM** 

### C **ITRATE** A 0.5 M sodium citrate solution was prepared by d issolving 1 47 .05 g of sodium citrate in 1000 mL of DI water. 

### 3.7. **P REPARATION OF**  

### D **ILUENT** The diluent was prepared by m ixing 800 mLo f 0.5 M citric acid with 200 mLo f 0.5 M sodium citrate.

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 3.8. **P REPARATION OF**  

### D **ERIVA TIZING**  

### A **GENT** 

### S **OLUTION** The deriv atizing agent, 2,4

- dinitropheny lhy drazine (DNPH), was prepared at a concentration of 3.00 mg/mL by w eighing approximately 0 .107 g of 2,4

- DNPH (30% water) in a tared glass weigh boat. The deri v atizing agent was transfer red to a 10

- mLv olumetric flask with rinses of ACN

. The preparation was mixed as necessary t o ach ieve complete dissolution of the deriv atizing agent. A dditional ACN was added to obtain the desired concentration, and the solution was thoroughly mixed. 

### 3.9. **P REPARATION OF**  

### C **ALIBRATION** 

### S **TOCK** 

### S **OLUTIONS** The calibration stock solution was prepared at a concentration of 5.00 mg acetaldeh yde/mL as follows. Approximately 0 .05032 g of acetaldehy de (WIL ID no. 110159; density

0 .788g/mL, no correction for purity) was accuratel y weighed into a tared gl ass container containing 10.0 mLo f DIw ater. A dditional DIw ater was added to achieve the desired concentration , and the solution was mixed with vortex action and/or gentle swirling. A secondary

c alibration standard stock solution was prepared at a concentration of 0.500 mg acetaldeh yde/ mLb y diluting an aliquot of the calibration stock solution 10

- fold with DI water. 

### 3.10. **P REPARATION OF**  

### C **ALIBRATION** 

### S **TANDARDS** Calibration standards at concentrations ranging from 10.0 to 50.0 μga cetaldeh yde/mL were prepared b y diluting aliquots of the secondary calibration stock solution with diluent in amber autosampler vials. A 450

- μL aliquot of the deriv atizing 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 standard s were prepared in triplicate at each concentration for the validation sessions; at least single calibration standards were prepared at each concentration for routine anal yses.

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 3.11. **P REPARATION OF**  

### Q **UALITY** 

### C **ONTROL** 

### S **TOCK** 

### S **OLUTION** A QC stock solution was prepared at a concentration of 5.00 mg acetaldehy de/mLa s follows. Approximately 0 .05032 g of acetaldehy de (WIL ID n o. 110159; density of 0.788g/mL , no correction for purit y) w as accurately w eighed into a tared glass container containing 10.0 mLof DIw ater. Add itional DIwater was added to achieve the desired concentrati on and the solution was mixed with vortex action and/or gentle swirling. 

### 3.12. **P REPARATION OF**  

### Q **UALITY** 

### C **ONTROL** 

### S **AMPLES** As detailed in the following table, QC samples were prepared to simulate the proce ssing of formulations at acetaldeh yde concentrations of 1.00, 3.00, and 5.00 mg acetaldehy de/mL (nominal QC concentration) by c ombining aliquots of the QC stock solution, vehicle (DIwater), 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 deriv atizing 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 Theoretic al

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. 8 0 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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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 3.13. **F ORMULATION**  

### S **AMPLE** 

### P **REPARATION** Duplicate formulation samples were prepared at a concentration of 5 mg acetaldeh yde/mL as follows. Approximately 2 .05 g of acetaldehy de (WIL ID no . 110159, densit y 0.788 g/mL, no correction for purit y) was accuratel y weighed into a tar ed glass container or a tared drinking water bottle containing approximately 5 0.0 and 100 mLo f DIw ater, respectively

. T he containers were brought to a final volume of 400 mLand thoroughly

m ixed. A dditional DIw ater was added to achieve the desired fi nal concentration. Duplicate formulation samples were prepared at a concentration of 1 mg acetaldehy de/mL by d iluted the 5 mg acetaldehy de/mL formulations 5

- fold with DI water. The preparations were thoroughl y mixed. 

### 3.14. **F ORMULATION**  

### S **AMPLE** 

### C **OLLECTION AND** 

### P **RO CESSING**  Quadruplicate samples were collected from each formulation using a sy ringe and dosing cannula and placed in glass tubes. T wo samples from each quadruplicate set were processed for anal ys is, and the remaining 2 samples (back

- up samples) were stored at room temperature and, if not needed for anal ys is, discarded after receipt of the Study Director’s approval of the analy tical results. A s detailed in the following table, formulation samples were processed by a dding diluent and mixing with vortex actio n. A portion of each formulation sample was further diluted with diluent in autosampler vials as necessary

t o achieve a theoretical final test substance concentration within the calibration concentration range. A 4 50

- μL aliquot of the deriv atizing 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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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. Group Target Test Substance

Conc entration (mg/mL) Sample

Amount (mL ) Diluent Volume (mL ) Secondary Dilution Theoret ical Final

Conc entration (μg/mL) Low 1 1.0 4.00 10

- fold 20.0 High 5 1.0 19.0 10

- fold 2 5 .0 NA = Not applicable 

### 3.15. **C ONCENTRATION**  

### Q **UANTITATION** Single injections were made of each processed calibration, QC, and formulation sample. A calibration curve was constructed for each set of analy ses. T he acetaldeh yde peak areas (y) and the theoretical concentrations (x) of the calibration standards were fit with least

- squares regression analy sis to the linear function with 1/x weighting: y = ax + b Concentrat ions were calculated from the results of the regression analy sis using Dionex Chromeleon ® software. The concentration data were transferred to a Microsoft Excel ® spreadsheet, where appropriate summary s tatistics, i.e. , mean, standard deviation (SD), relat ive standard deviation (RSD), percent relative error (%RE), and concentration as a percent of target, were calculated and presented in tabular form. T he concentrations of the formulation and QC samples were calculated by a pply ing any

n ecessary multiplicat ion factors to correct for dilution and/or unit conversions. 

### 3.16. **WIL R** **ESEARCH** 

### C **OMPUTER** 

### S **YSTEMS** 

### 3.16.1. **R EPORTING**  **AND** 

### A **NCILLARY** 

### S **YSTEMS** 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. ) InSi ght ® 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. **R ESUL** 

**TS AND**  

### D **ISCUSSION** Under the described chromatographic conditions, the retention time of the test substance was approximately

5 .2 minutes. F igure 1 , Figure 2, Figure 3 , Figure 4 , and Figure 5 are ty pical chromatograms of a calibration standard, a processed QC sample, a processed formulation sample, a processed vehicle blank sample, and a processed diluent sample, respectivel y. The total analy sis time required for each run was 15 minutes.

0. 0 2. 0 4. 0 6.0 8. 0 10. 0 12 .0 15. 0 -5 00 1, 000 2, 000 3, 500 LC 7- 876002H #10 R un # 301 876002- 90- 1 UV _V IS _1 m A U

m i n **1 -**  **Peak 1 -** 

**5 .1**  **53** WV L: 360 nm

Figure 1: Representative Chromatogram of a 10.0 μg A cetaldehy de/mLCalibration

Standard

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc.

0. 0 2. 0 4. 0 6.0 8. 0 10. 0 12 .0 15. 0 -5 00 1, 000 2, 000 3, 500 LC 7- 876002H #34 R un # 325 876002- 93- 8 UV _V IS _1 m A U

m i n **1 -**  **Peak 1 -** 

**5 .1**  **63** WV L: 360 nm

Figure 2: Representative Chrom atogram of a Processed 5.00 mg A cetaldeh yd e/mL

Quality Control Sample

0. 0 2. 0 4. 0 6.0 8. 0 10. 0 12 .0 15. 0 -5 00 1, 000 2, 000 3, 500 LC 7- 876002H #59 R un # 350 876002- 98- 3 UV _V IS _1 m A U

m i n **1 -**  **Peak 1 -** 

**5 .1**  **69** WV L: 360 nm

Figure 3: Representative Chromatogram of a Processed 5. 00 mg A cet aldeh yd e/mL

Formulation Sample **Page 17 of 60**WI L

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc.

0.0 2. 0 4. 0 6. 0 8. 0 10. 0 12 .0 15. 0 -1 5 20 40 60 80 100 LC7 -8 76 002 H #2 7 R un # 3 18 876002- 93- 1 UV _V IS _1 m A U

m i n **1 -**  **Peak 1 -** 

**5 .0**  **49** WV L: 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 R un # 316 Dil 876002- 82- 1 UV _V IS _1 m A U

m i n WV L: 360 nm

Figure 5: Chromatogram of a Processed Diluent Sample

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 4.1. **S PECIFICITY** 

### /S **ELECTIVITY** 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 a ffect the quantitation of the test substance

- containing formulations. As shown in Figure 5 , anal ys is of processed diluen t 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. **A SSAY**  

### V **ALIDATION** 

### : **C ALIBRATION**  

### R**EP RODUCIBILITY**  During each of the 3 method va lidation sessions, triplicate calibration standards at 5 concentrations were prepared and anal yz ed as described previousl y. S ingle injections were made of each calibration standard. T he resulting acetaldeh yd e peak area versus theoretical acetaldeh yde co ncentration data were fit to the linear function using least

- squares regression anal ys is. T he results of the regression analy ses were used to back

- calculate the corresponding concentrations from the peak area data. As per protocol criteria, the reproduc ibility of the calibration curve data was considered valid when 1) the inter

- session variabilit y, expressed as RSD, of the back

- calculated concentrations at each calibration level was ≤10%, except at the lowest calibration level where ≤15% was a cceptable; 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 acetaldeh yd e assay c alibration standards are summarized in Table 1

. T he inter

- session variability (RSD) of the back

- calculated c oncentrations ranged from 0. 54 % to 2.3% RSD. The inter

- session mean concentrations had %RE values ranging from

- 0. 30 % to 0. 16 %. B ased on the stated criteria, the reproducibility o f the calibration data was acceptable.

**Page 19 of 60**WI L

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 4.3. **A SSAY**  

### V **ALIDATION** 

### : **P RECISION AND**  

### A **CCURACY** During each of 3 validation sessions , triplicate QC samples at 3 concentrations were prepared and anal yz ed as described previously

. S ingle injections were made of each processed QC sample. T he results of the regression anal ys es were used to calculate the corresponding concentrations from the QC peak area data. T he variability (RSD) of the calculated QC concentration data was used as a measure of assay p recision, and the difference between theoretical and the calculated mean QC concentrations (%RE) was used as a measure of assay a ccuracy . 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 o f the method was considered acceptable when the inter

- session calculate d 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 acetaldeh yde assay

Q C samples are summarized in Table 2

. The inter

- session variability (RSD) of the calculat ed concentrations of each QC sample (precision) ranged from 5. 7 % to 7.9% RSD. T he inter

- session mean concentrations of the QC samples had %RE values (accuracy) ranging from

- 5.0 % to
- 3. 4 %. B ased on the stated criteria, the precision and accuracy

of the a cetaldeh yde assay were acceptable. 

### 4.4. **A SSAY**  

### R**UGGEDNESS** Assay r uggedness, as required by WI L Research SOP, was successfull y demonstrated for this method because at least 2 of the 3 validation sessions were performed by d ifferent anal ys ts. 

### 4.5. **A SSAY**  

### A **CCEPTABILI TY**  In addition to the experimental samples, each analy tical 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 prepar ed at target ac etaldehy de concentrations of 1 and 5 mg/mL , and the QC samples were prepared at **Page 20 of 60**WI L

- 876002 Acetaldeh yd e

Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. nominal acetaldeh yde concentrations of 1.00, 3.00 , and 5.00 mg/mL. F or an anal yt ical session to be considered valid, at least two
- thirds of the calculated QC co ncentrations with at least 1 sample at each concentration had to be 90% to 110% of the nominal QC concentration. A ll reported results were from analy tical sessions that met the acceptance criteria. 

### 4.6. **T EST**  

### S **UBSTANCE** 

### S **TABILITY IN** 

### C **ALIBRATION** 

### S **TANDARDS** Calibration standards prepared at 10.0 and 50.0 μg/mL and anal yz ed on 14 December 2011 were stored at room temperature for a minimum of 5 day s before being re - anal yz ed to assess test substance stability

. T he mean post
- storage concentrations were 90.8 % and 99.5 % of the pre - storage values ( Table 3 ), which met the protocol

- speci fied requirement for stability , i.e. , the mean post
- storage concentration was not < 90% of the pre

- storage value

. 

### 4.7. **T EST**  

### S **UBSTANCE** 

### S **TABILITY IN** 

### P **ROCESSED** 

### S **AMPLES** QC samples prepared at nominal test substance concentrations of 1.00 and 5.00 mg/mL were processed and analyzed on 14 December 2011. T he processed samples were stored at room temperature for a minimum of 5 day s before being re - anal yz ed to assess test substance stability

. T he mean pos t
- storage concentrations were 97.9 % and 99. 2 % of the pre - storage values ( Table 3 ), which met the previously s tated protocol

- specified requirement for stability

. 

### 4.8. **T EST**  

### S **UBSTANCE** 

### T **IME** 

### - **Z ERO**  

### C **ONCENTRATION** 

### A **SSESSMENT OF** 

### F **ORMULATIONS** Duplicate samples from the mi ddle strata of formulations prepared on 19 December 2011 at target concentrations of 1 and 5 mg acetaldehy de /mL were analyzed to verify t est substance concentration acceptability i n both glass containers and drinking water bottles. The results of the time

- zero concentration acceptability a ssessments are presented in Table 4 , with the mean concentrations and overall statistics summarized in the following table.
**Page 21 of 60**WI L

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Acetaldeh yd e Working Group of the Vin yl 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 Targ et 92.1 95.7 93.4 104 The anal yz ed formulations met the protocol

- specified requirement for time

- zero concentration acceptability f or DIw ater formulations, i.e. , the analyzed concentration was 90% to 110% of the target concentration. 

### 4.9. **T EST**  

### S **UBSTANCE** 

### S **TABI LITY OF**  

### F **ORMULATIONS IN** 

### G **LASS** 

### C **ONTAINERS** The formulations prepared and anal yz ed on 19 December 2011 were stored in glass containers at room temperature for 24, 48 , and 72 hours before being re - anal yz ed to assess test substance stability

. T he results of th e stability ana ly sis are presented in Table 5 , Table 6, and Table 7

. T he 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 previousl y stated protocol

- specified requirement for stability w ith the following exception. The low group (1 mg/mL) formulation prepared

**Page 22 of 60**WI L

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

- z ero concentration which did not meet the stated criteria for stabilit y. 

### 4.10. **T EST**  

### S **UBSTANCE** 

### C **OMPATIBILITY OF** 

### F **ORMULATIONS IN** 

### D **RINKING** 

### W **ATER** 

### B **OTTLES** The formulations prepared and anal yz ed 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 - anal yz ed to assess test substance compatibility

. The results of the compatibility ana ly sis are presented in Table 8 , Table 9 , and Table 10

. The mean conc entrations and percent of post
- storage concentration in glass container s are summarized in the follo wing table. Mean Concentration, mg/mL (% of Post

- Storage Concentration in Glass Containers ) Storage Condition Storage Duration Low Group High Grou p (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 1 13% of the glass container post
- storage values, which met the protocol

- specified requirement for compatibility , i.e. , the post
- storage assay c oncentration was not < 95% of the post
- storage concentration of the formulation stored in glass containers.

**Page 23 of 60**WI L

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 5. **C ONCLUSION**  **S** A validated HPL C/UV me thod for the analy sis of acetaldehy de concentration in DI water formulations and test substance ranging in concentration from 1 .00 to 5 .00 mg/mLw as validated in this study. A ssay s pecificity

/selectivity , calibration reproducibility , precision, accuracy , ruggedness, and test substance stability i n calibration standards and processed QC samples stored at room temperature for a minimum of 5 day s were assessed and validated, satisfy ing protocol

- specified criteria. Formulations prepared at target test substanc e concentrations of 1 and 5 mg acetaldeh yde/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 acetaldeh yde/ mLm et the protocol

- specified requirement for formulation compatibility

w ith 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 60WIL-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 60WI L

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 8. **D ATA**  

### R**ETENTION** The raw data, the retention sample(s) if applicable, pertinent electroni c storage media, and the original final report are retained in the WI L Research Archives in compliance with regulatory

requirements.

**Page 27 of 60**WI L

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Acetaldeh yd e Working Group of the Vin yl Acetate Council, Inc. 

### 9. **A BBREVIA** 

**TIONS** The following abbreviations may apply to this report: μ - micro μ L

- micro liter ACN

- a cetonitrile btm

- bottom conc. - concentration DI

- d eionized ESI +
- p ositive electrospray ionization FA

- f ormic acid FDA

- Food and Drug Administration g - gram GL P

- Good Laboratory Practices HPL C

- h igh performance liquid chromatograph y hr

- hour(s) IS

- in ternal standard kg

- kilogram L

- liter LLOQ

- l ower limit of quantitation MC

- m ethy lcellulose mg - milligram mL

- milliliter mm - millim e ter ms ec - milliseconds MS

- m ass 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

- p ercent relative error RSD

- r elative standard deviation SD

- s tandard deviation SOP

- standard operating procedure UV

- u ltraviolet v - volume w - weight WI L Research

- WI L Research Laboratories, LLC
**Page 28 of 60**WI L

- 876002 Acetaldeh yd e

Acetaldeh yd e Working Group of the Vin yl 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 D rinking Bottle Formulations Low Mi d 1 61 - 1 162 0.941 94.1 0.934 0.0098 1.1 93.4 61 - 2 163 0.927 92.7 Hi gh Mi d 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 - )WIL-876002 AcetaldehydeAcetaldehyde Working Group of theVinyl 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

Grou p Lo w Hig h 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 4.78WIL-876002 AcetaldehydeAcetaldehyde Working Group of theVinyl 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

Grou p Lo w Hig h 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 4.78WIL-876002 AcetaldehydeAcetaldehyde Working Group of theVinyl 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

Grou p Lo w Hig h 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)

0.921WIL-876002 AcetaldehydeAcetaldehyde Working Group of theVinyl 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 4.49 5.20WIL-876002 AcetaldehydeAcetaldehyde Working Group of theVinyl 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 4.59WIL-876002 AcetaldehydeAcetaldehyde Working Group of theVinyl 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)WIL-876002 AcetaldehydeAcetaldehyde Working Group of theVinyl 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. 

### D EVIATIONS F ROM T HE 

### P ROTOCOL 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 st orage. 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 w ere 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 PERSOmLINVOLVED 

### 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.

**Page 52 of 60**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 **Page 53 of 60**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 **Page 54 of 60**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 **Page 55 of 60**WIL-876002 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 **Page 56 of 60**WIL-876002 Page 10 of 11 November 7,2011 

### 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.

**Page 57 of 60**WIL-876002 Page 11 of 11 November 7,201 1 

### 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

**Page 58 of 60**WIL-876002 Acetaldehyde Acetaldehyde Working Group of the Vinyl Acetate Council, Inc.



### APPENDIX B Certificate of Analysis

**Page 59 of 60 Page 60 of 60**