## CiToxLAB Hungary Ltd.

Veszprém

- Szabadságpuszta



# FINAL REPORT

Determination of the Water Solubility

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

- 56

- 6]

(hydrated form of ethanedioic acid [CAS RN 144

- 62

- 7])

Study code: 11/ 3 39

- 345AN

Study Director: Zsolt Sárvári, M.Sc.

20 February 201 3Study code: 11/ 3 39

- 345AN Final Report Page 5 of 16

GENERAL INFORMATION

STUDY TITLE : Determination of the Water Solubility 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 11 021, 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 635

Palm Beach Gardens, FL 33410, USA

Phone: +1 561 578 8110

Fax: +1 561 578 8120

E

- m ail: john@grcci.com

TEST FACILITY: CiToxLAB Hungary Ltd.

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.

Sen ior Director of Toxicology Operations

STUDY DIRECTOR: Zsolt Sárvári M.Sc.

Phone: +36 88 545 361

E

- mail: zsolt.sarvari @hu.citoxlab.comStudy code: 11/ 3 39

- 345AN Final Report Page 6 of 16

ASSISTANT SCIENTIST : Olga Ulbert , M.Sc.

LEAD QA : Szabolcs Gáty M.Sc.

Head of Quality Assurance Unit

Vanda Gyi mesi, M.Sc.

Deputy Head of QAU

RESPONSIBLE PERSONS: Ildikó Kacz

- technical assistant

BASIS OF STUDY:

- Commission Regulation (EC) No 440/2008 of 30 May

2008 laying down test methods pursuant to Regulation

(EC) No 1907/2006 of the European Parliamen t and of the

Council on the Registration, Evaluation, Authorisation and

Restriction of Chemicals (REACH), Part A, A.6 “ Water

Solubility ”, Official Journal of the European Union L 142

of 31 May

- Organisation for Economic Co

- operation and

Development (OECD ), OECD Guidelines for Testing of

Chemicals, Guideline No. 105, "Water Solubility",

adopted July 27, 1995Study code: 11/ 3 39

- 345AN Final Report Page 7 of 16



### CONTENTS

Page

1. OBJECTIVE OF STUDY 8

2. MATERIALS 8

2 .1. Test Item 8

2.2. Reagents and Materials 9

3. TEST METHODS AND RES ULTS 9

3.1. Principle of the Test 9

3.2. Apparatus 9

3.3. Results of the Method Validation 10

3.4. PERFORMANCE OF THE TEST 10

3.5. results 11

3.6. conclusion 12

4. A RCHIVES 12

5. DISTRIBUTION OF THE FINAL REPORT 13

APPENDIX 1 4

Copy of the GLP Certificate 15

Certificate of Analysis 16Study code: 11/ 3 39

- 345AN Final Report Page 8 of 16

1. OBJECTIVE OF STUDY

This study was performed based on the U.S. Toxic Substances Control Act (TSCA)

Section 4 Test Rule and the regulations set forth at 40 CFR 799.5087.

The purpose of this study wa s to determine the water solubility of Ethanedioic acid,

hydrate (1:2) [CAS RN 6153

- 56

- 6] (hydrated form of ethanedioic a cid [CAS RN 144

-

62

- 7]) which is specified by the saturation mass concentration of the test item in water

at 20 **±** 1 °C

.

2

. MATERIALS

2 .1. TEST ITEM

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

- 56

- 6] (hydrated

form of ethanedioic acid [CAS RN 14 4

- 62

- 7])

Chemical name: Oxalic acid, hydrate

CAS number: 6153

- 56

- 6

Batch/Lot number: YP1

- 20120528C

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

Molecular weight: 126.1 (dihydrate form)

Appearance: White crystals

Purity: 99.69 %

Manufacture date: May 28, 2012

Expiry dat e: May 27, 2013

Storage conditions: Controlled room temperature (15

- 25 o C)

2 .1.1. Identification, 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.Study code: 11/ 3 39

- 345AN Final Report Page 9 of 16

2

. 2

. REAGENTS AND MATERIA LS

Ultra pure water (ASTM Type I): prepared by D irect

- Q 3 system, Millipore

Acetonitrile : HPLC grade, VWR , Batch: 11Z3738

Potassium dihydrogen phosphate: reagent, Lach

- Ner, Batch: PP/2010/02241

Phosphoric acid 85%: reagent, Lach

- Ner, Batch: PP/2011/12163

3

. TEST METHODS AND RES ULTS

Start of experi ment: 20 November 20 1 2

End of experiment: 24 November 20 1 2

3.1. PRINCIPLE OF THE TES T

The substance is dissolved in water at a temperature somewhat above the test

temperature. When saturation is achieved the mixture is cooled and kept at the test

temp erature, stirring as long as necessary to reach equilibrium. Subsequently, the mass

concentration of the substance in the aqueous solution, which must not contain any

undissolved particles, is determined by a suitable analytical method.

3

. 2

. 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

- 033

L

- 7400 UV Detector, No.: 1374

- 025

L

- 7614 Degasser, No.: 144116A0094

L

- 7360 Column Oven, No.: 00118060

Ba lance s : BP221S 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, No.:1231025119

Refrigerator: Zanussi, No.: ZLKI

- 262

Incubator: Gallencamp, No.: 8B3262JStudy code: 11/ 3 39

- 345AN Final Report Page 10 of 16

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: 10 μl

Temperature: 25 °C

Detector: UV at 2 2 0 nm

Ret ention time: 2

. 5 min ± 10%

3.3. RESULTS OF THE METHO D VALIDATION

Table 1.: Results of the Method Validation (Study Code: 11 / 3 39

- 316AN)

Selectivity No interfering component was observed

Reinjection repeatability

( 7 injections) CV% ≤ 1

. 6 %

Linear range 1

– 10 0 μg/ml

Limit of Quantification 1 μg/ml

Recovery from water 106 %

Stability of the samples At least 19 hours in the autosampler

Stock solution stability At least 4 days at 5 **±** 3 °C

3 .4. PERFORMANCE OF THE T EST

About 30 g test item measured into 10 0 ml stoppered Erlenmeyer flasks (nine parallel)

. After the addition of 5 0 ml ultra pure water, the flasks were agitated at 30 °C.

After one day, three of the vessels were removed and re

- equilibrated at 20 °C for 24

hours. The concentration in the clear solvent phase was determined by HPLC.

The other flasks were treated similarly after initial equilibration at 30 °C for two and

three days, respectively. The samples were filtered with 0.22 μm filter and diluted

before the analysis (two parallel dil utions and two injections each).

Every occasion control water is measured.

The pH of each sample was recorded.Study code: 11/ 3 39

- 345AN Final Report Page 11 of 16

3. 5

. RESULTS

3

. 5 .1. Calibration

Calibration samples were prepared from the analytical standard. Seven c oncentrations

were used: 1, 2, 5

, 1 0

, 20

, 50 and 10 0 μg/ml . The calibration graph was calculated

applying linear regression

. Parameters of the line are presented in Table 2 ; plot of the calibration graph can be

seen in Figure 1.

Table 2 : Summary of Standard Curve Parameters

Date Intercept Slope Correlation Coefficient

22 November 2012 486 2964 1.000

23 November 2012 644 2791 1.000

24 November 2012 63 2610 1.000

0 50000 100000 150000 200000 250000

0 20 40 60 80 100 120Area

Concentration (μg/ml)

Figure 1: Linearity test on 2 4 November 201 2

3

. 5

. 2

. Visual observation

Test item proved to be soluble with water. The t est mixtures were clean, viscous

.Study code: 11/ 3 39

- 345AN Final Report Page 12 of 16

3

. 5

. 3

. Analysis of the samples

pH of the samples varied in the range of 0

. 58

- 0

. 80

.

S amples were analysed with the above HPLC method.

Measured concentrations are summarised in Table 3

.

Table 3 : Results of the deter mination of test item concentrations

Measured

concentration

after 24 hours

( m g/ m L) pH Measured

concentration

after 48 hours

( m g/ m L) pH Measured

concentration

after 72 hours

( m g/ m L) pH Solubility of

the test item

( m g/ m L)

119 0.80 131 0.77 137 0.60

129 **±** 6 .0 120 0.75 128 0.69 136 0.59

120 0.76 128 0.62 139 0.58

Mean: 120 **±** 2.4

- Mean: 128 **±** 5 .2

- Mean: 139 **±** 2.9

- RSD%= 6

3. 6

. CONCLUSION

In conclusion, the water solubility of Ethanedioic acid, hydrate (1:2) [CAS RN 6153

-

56

- 6] (hydrated form of etha nedioic acid [CAS RN 144

- 62

- 7]) was determined to be

129 **±** 6

. 0 m g/ m L (95% confidence interval) at 20 °C ± 1°C using the shake

- flask

method for the perfor m ance of the main test.

4

. ARCHIVES

The study documents:

- study plan and amendments

- all raw dat a

- sample of the test item

- study report and any amendment(s)

- correspondence

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

Szabadságpuszta, Hungary, according to the Hungarian GLP and our SOPs.Study code: 11/ 3 39

- 345AN Final Report Page 13 of 16

5

. DISTRIBUTION OF THE FINAL REP ORT

Sponsor:

- 1 × copy, bound

- 1 × copy, unbound

- 1 × pdf

- file

Archives:

- 1 × original, boundStudy code: 11/ 3 39

- 345AN Final Report Page 14 of 16



## APPENDIXStudy code: 11/ 3 39

- 345AN Final Report Page 15 of 16



### Copy of the GLP CertificateStudy code: 11/ 3 39

- 345AN Final Report Page 16 of 16



### Certificate of Analysis