## CiToxLAB Hungary Ltd.

Veszprém

- Szabadságpuszta



# FINAL REPORT

Validation of the Analytical Method for the

Determination of Ethanedioic acid, hydrate (1:2) [CAS

RN 6153

- 56

- 6] (hydrated form of ethanedioic acid [CAS

RN 144

- 62

- 7])

Study code: 11 / 339

- 316AN

Study Director: Zsolt Sárvári , M.Sc.

2 1 February 201 3Study code: 11 / 339

- 316AN Final Report Page 5 of 18

STUDY TITLE: Validation of the Analytical Method for t he Determination

of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6]

(hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

SPONSOR S : Univar USA Inc.

Address: 17425 NE Union Hill Road,

Redmond, WA 98052, USA

Phone: +1 425 889 3400

Fax: +1 425 889 3760

WEGO Chemical and Mineral Corp.

Address: 239 Great Neck Road,

Great Neck, NY 11021, USA

Phone: + 1 516 487 3510

Fax: +1 516 487 3794

STUDY MONITOR: John V. Massingale

Greenwich Chemical Consulting, Inc.

Address: 3300 PGA Boulevard, Suite 63 5

Palm Beach Gardens, FL 33410, USA

Phone: +1 561 578 8110

Fax: +1 561 578 8120

E

- mail: john@grcci.com

TEST FACILITY : CiToxLAB Hungary Ltd.

(Formerly known as LAB Research Ltd. before 1 st September 2011)

Address: H

- 8200 Veszprém, Szabadságpuszta

Hungary

Phone: +36 88

- 545

- 300

Fax: +36 88

- 545

- 301

TEST FACILITY

MANAGEMENT: Christopher Banks, DABT

Managing Director

Alyson Leyshon, M.Sc.

Senior Director of Toxicology Operations

STUDY DIRECTOR: Zsolt Sárvári M.Sc.

Phone: +36 88 545 361

E

- mail: zsolt.sarvari @hu.citoxlab.comStudy code: 11 / 339

- 316AN Final Report Page 6 of 18

ASSISTANT SCIENTIST : Olga Ulbert , M.Sc.

LEAD QA : Szabolcs Gáty M.Sc.

Head of Quality Assurance Unit

Vanda Gyimesi, M.Sc.

Deputy Head of QAU

RESPONSIBLE PERSONS: Ildikó Kacz

- technical assistant

Andrea Egei

- technical assistant

BASIS OF STUDY: SOP of CiTox LAB Hungary Ltd.; ANE 124,

"Validation of Analytical Method"Study code: 11 / 339

- 316AN Final Report Page 7 of 18



### CONTENTS

Page

1. SUMMARY 8

2. MATERIALS 9

2.1. TEST ITEM 9

2.2. REAGENTS AND MATERIA LS 9

2.3. MIXTURES, SOLUTIONS 9

3. TEST METHODS AND RES ULTS 10

3.1. APPARATUS 10

3.2. ANALYTICAL METHOD 10

3.3. METHOD VALIDATION 11

4. ARCHIVES 14

5. DISTRIBUTION OF THE FINAL REPORT 14

6. DEVIATION 14

APPENDIX 1 5

Copy of the GLP

- Certificate s 1 6

Certificate of Analysis 1 8Study code: 11 / 339

- 316AN Final Report Page 9 of 18

2. MATERIALS

2.1. TEST ITEM

Name: Ethanedioic acid, hydrate (1:2) [CAS RN 6153

- 56

- 6]

(hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

Chemical name: Oxalic acid, hydrate

CAS number: 6153

- 56

- 6

Batch/Lot number: YP2

- 20110704B

Molecular formula: C 2 H 2 O 4 ×2H 2 O

Molecular weight: 126.1 (dihydrate form)

Appearance: White crystals

Purity: 99.71 %

Manufacture date: 04 July 2011

Expi ry date: 03 July 2012

Storage conditions: Controlled room temperature (15

- 25 o C)

2.1.1. Receipt

The test item of a suitable chemical purity was supplied by the Sponsor. All safety

instructions required in the handling and disposal of the test item were supplied by

the Sponsor as well.

Identification of the test item was made by its name, batch, appearance and colour by

Central Dispensary Unit of CiToxLAB Hungary Ltd.

2.2. REAGENTS AND MATERIA LS

Ultra pure water (ASTM Type I): prepared by Direct

- Q 3 s ystem, Millipore

Acetonitrile : HPLC grade, VWR , Batch: 12Z0028

Potassium dihydrogen phosphate: reagent, Lach

- Ner, Batch: PP/2010/02241

Phosphoric acid 85%: reagent, Reanal, Batch: KBM55463

2.3. MIXTURES, SOLUTIONS

Stock Solution: 1

. 0 mg Ethanedioic acid / 1 ml water

Calibration series: 1

, 2

, 5

, 10, 20, 50 and 1 0 0 μg test item / ml: Stock

Solution and 5 0 μg/ml working solution w ere properly

diluted with mobile phase

.Study code: 11 / 339

- 316AN Final Report Page 10 of 18

Recovery Formulations: 100 mg/l: About 1 0 mg of test item was measured into

10 0 ml flask s (one decimal place accuracy). It was

dissolved with 10 0 ml matrix

.

5 mg/ml: 5 ml 100 mg/ml solution measured into the

100 ml flask and diluted with 100 ml matrix

.

3. TEST METHODS AND RES ULTS

Start of experiment: 14 May 201 2

End of experiment: 25 May 201 2

3.1. APPARATUS

HPLC system: Merck

- Hitachi LaChrom HPLC system

D

- 7000 Interface, No.: 1126

- 175

L

- 7100 HPLC pump, No. : 1388

- 056

L

- 7200 Autosampler, No.: 1380

- 0 33

L

- 7400 UV Detector, No.: 1374

- 0 25

L

- 761 4 Degasser, No.: 144116A0094

L

- 7360 C olumn Oven, No.: 00118060

Balance s : BP2 21 S Sartorius, No.: 11809117

L2200P Sartorius, No.:3810037

Ultrasonic bath: Elmasonic S300H, ELMA, No.: 010890105

Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I

pH meter: SevenEasy S20 Mettler Toledo, N o.:1231025119

Refrigerator: Zanussi, No.: ZLKI

- 262

3.2. ANALYTICAL METHOD

3.2.1. HPLC Conditions:

Column: Zorbax SB

- C 8 2 50 × 4.6 mm, 5μm

Mobil Phase : 50 mM KH 2 PO 4 (pH:2.1) : ACN 9 : 1

Flow: 1.0 ml/min.

Injection volume: 1 0 μlStudy code: 11 / 339

- 316AN Final Report Page 11 of 18

Temperature: 25 °C

Dete ctor: UV at 2 2 0 nm

Retention time: 2

. 5 min ± 10%

3.3. METHOD VALIDATION

The following characteristics were determined:

3.3.1. Selectivity

For the selectivity test the aqueous control matrices were analysed.

No interfering peak was observed on the blank chromatograms.

3.3.2. Repeatability

Re

- injection repeatability was tested with 7 consecutive injections of the 2 and 50

μg/ml calibration standards on two different days . CV of the replicate injections

was determined. The repeatability was found to be 1

. 6 % or better.

Table 2.: The injection repeatability of the method

Sample Measured

parameter RSD (CV) %

Analytical

occasion 1. Analytical

occasion 2.

Standard 2 μg/ml Retention time 0.2 0. 0

Area 1.6 0.8

Standard 50 μg/ml Retention time 0.0 0. 2

Area 0.4 0.7

3.3.3. Linearity

Linearity of the HPLC method was examined in 1

- 1 0 0 μg/ml range. Seven

concentration levels were tested: 1

, 2

, 5

, 10, 20, 50 and 1 0 0 μg/ml.

Five such series were prepared from separate stock solutions and analysed on five

different days.

The detector response proved to be linear in 1

– 1 0 0 μg/ml range. The calibration

graph was calculated applying weighted linear regression using 1/concentration as

weighting factor.Study code: 11 / 339

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Deviation of the back

- calculated values was le ss than 15 %.

Parameters of the curves are presented in Table 3; plot of the calibration graph can

be seen in Figure 1.

Table 3: Summary of Standard Curve Parameters

Analytical occasion Intercept Slope Correlation

Coefficient

14 May 201 2

- 286 2771 1.00 0

15 May 201 2 264 2617 1.000

17 May 201 2 108 2900 1.000

18 May 201 2

- 523 2861 1.000

21 May 201 2

- 468 2878 1.000

0 50000 100000 150000 200000 250000 300000

0,00 20,00 40,00 60,00 80,00 100,00 120,00Area

Concentration (μg/ml)

Figure 1: Linearity test on 21 May 201 2

3.3.4. Limit of Quantification

The lowest concentration of the calibration curve, 1 μg/ml is considered to be the

quantification limit of the HPLC method.Study code: 11 / 339

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3.3.5. Recovery, Accuracy and Precision

Recovery of test item was tested f ro m alga and daphnia test solution s and water

. Concentration of the test solutions was 5 and 100 mg/l and the wate r was 75 mg/ml

. Five replicate solutions were made.

The recovery samples were analysed by the above HPLC method. The results are

presented in Table 4.

Table 4 : Recovery, Accuracy and Precision of the analytical method

Matrix Nominal

concentration Recover y

% Accuracy

% Precision

%

Alga test solution 5 mg/l 105 5 2

100 mg/l 100 0 1

Da ph nia test solution 5 mg/l 105 5 2

100 mg/l 101 1 3

W ater 75 mg/ml 106 6 4

3.3.6. Stability

Stock solution stability

Stock solution (1 mg/ml) prepared in water pr oved to be stable for at least 4 days at

5 **±** 3 °C. :

Peak area of the 1 0 μg/ml solution prepared from the 4 days old stock solution was

100 % of the peak area of the fresh one.

Stability of the samples in the autosampler

Calibration samples ( 2 and 5 0 μg/m l) were reinjected after 19 hours storage period

in the autosampler. Peak areas after the storage period were 9 4 and 98 % of the

areas measured at the start. :

Stability of the test item in test solutions

Test solutions and water sample w as stored at room temperature. At the end of the

storage period formulations were analysed. :

The results are detailed in Table 5.Study code: 11 / 339

- 316AN Final Report Page 14 of 18

Table 5: Results of the stability test in aqueous solutions

Matrix Nominal

concentration Storage

period End/Start

Alga test solution 5 mg/l 3 day 94 %

100 mg/l 3 day 100 %

Daphnia test solution 5 mg/l 2 day 89 %

100 mg/l 2 day 100 %

Daphnia test solution

for fish test 5 mg/l 4 day 112 %

100 mg/l 4 day 115 %

Water 75 mg/ m l 3 day 97 %

4. ARCHIVES

The study documents:

- study plan

, amendment

- all raw data

- sample of the test item

- study report and any amendment(s)

- correspondence

are stored in the archives of CiTox LAB Hungary Ltd., Hungary 8200 Veszprém,

Szabadságpuszta according to the Hungarian GLP and our SOP

- s.

5. DISTRIB UTION OF THE FINAL R EPORT

Sponsor:

- 1 × pdf

- file

Archives:

- 1 × original, bound

6. DEVIATION

The date of the Draft Report was 19 June 2012 instead of 15 June 2012 , because of

the internal scheduling . This deviation does not have any effect on study re sults.Study code: 11 / 339

- 316AN Final Report Page 15 of 18



## APPENDIXStudy code: 11 / 339

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### Copy of the GLP

- Certificate s

The facility name until 1 st September 2011 was LAB Research Ltd. From that date, the registered name has been CiToxLAB Hungary Ltd.,

this information has been transmitted to the GLP competent authority. The above GLP certificate is valid for this facility (n ow known as

CiToxLA B Hungary Ltd ) until the certificate expires (16 December 2012).Study code: 11 / 339

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### Certificate of Analysis