## CiToxLAB Hungary Ltd.

Veszprém

- Szabadságpuszta



# FINAL REPORT

Validation of the Analytical Method for the

Determination of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

-

Study code: 1 2 / 056

- 316AN

Study Director: Zsolt Sárvári , M.Sc.

12 March 201 3Study code: 1 2 / 056

- 316AN Final Report Page 5 of 18

STUDY TITLE: Validation of the Analytical Method for the Determination

of Benzene, 1

- chloro

- 4

- (trifluoromethyl)

-

SPONSOR: PCBTF Testing Consortium

Address: c/o Greenwich Chemical Consult ing, Inc.

3300 PGA Boulevard, Suite 635

Palm Beach Gardens, FL 33410

USA

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

STUDY DIRECTOR: Zsolt Sárvári, M.Sc.

ASSISTANT SCIENTIST: Olga Ulbert, M.Sc.

QUALITY ASSURANCE: Vanda Gyimesi, M.Sc.

On behalf of QAU

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

"Validation of Analytica l Method"Study code: 1 2 / 056

- 316AN Final Report Page 6 of 18



### CONTENTS

Page

1. SUMMARY 7

2. MATERIALS 8

2.1. TEST ITEM 8

2.2. REAGENTS AND MATERIA LS 8

2.3. MIXTURES, SOLUTIONS 8

3. TEST METHODS AND RES ULTS 9

3.1. APPARATUS 9

3.2. ANALYTICAL METHOD 10

3.3. METHOD VALIDATION 10

4. ARCHIVES 13

5. DISTRIBUTION OF THE FINAL REPORT 14

6. DEVIATION 14

APPENDIX 1 5

Copy of the GLP Certificate s 1 6

Certificate of Analys is 18Study code: 1 2 / 056

- 316AN Final Report Page 8 of 18

2. MATERIALS

2.1. TEST ITEM

Name: Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- Synonym: PCBTF

CAS Number: 98

- 56

- 6

Batch/Lo t number: 20111028

Molecular formula: C 7 H 4 ClF 3 Molecular weight: 180.5 6

Appearance: Clear colourless liquid

Purity: 99.69%

Manufacture date: 28 October 2011

Expiry date: 28 October 2013

Storage conditions: Room temperature

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 colo ur 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 system, Millipore

Acetonitrile (ACN) : HPLC grade, VWR , Batch: 12Z 3715

2.3. MIXTURES, SOLUTIONS

Stock Solution: 1

. 0 mg Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- / 1 ml

ACN

Calibration standards : 0. 1

, 0. 2

, 0. 5

, 1, 2, 5 , 10 and 2 0 μg Benzene, 1

- chloro

- 4

- (trifluoromethyl)

- / ml: Stock Solution and 1 0 μg/ml

working solution w ere properly diluted with ACN :

water = 7:3

.

Rec overy Formulations: 2 0 mg/l: About 1 0 mg of test item was measured into

5 0 0 ml flasks (one decimal place accuracy). It was

dissolved with 5 0 0 ml alga test solution

.

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

100 ml flask and diluted with 100 ml ma trix

.Study code: 1 2 / 056

- 316AN Final Report Page 9 of 18

3. TEST METHODS AND RES ULTS

Start of experiment: 16 October 201 2

End of experiment: 2 6 October 201 2

3.1. APPARATUS

HPLC system: Merck

- Hitachi LaChrom HPLC system III

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 Column Oven, No.: 00118060

Merck

- Hitachi LaChrom HPLC system I V

D

- 7000 Interface, No.: 1442

- 122

L

- 7100 HPLC pump, No. : 1516

- 030

L

- 7200 Autosample r, No.: 1406

- 005

L

- 7400 UV Detector, No.: 1502

- 017

L

- 7614 Degasser, No.: 14412YA0500

L

- 7360 Column Oven, No.: 00107295

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, No.:1231025119

Shaking incubator: Gallencamp 8B 3262 J

Spectrop hotometer: Hitachi U

- 2910, No.: 2221

- 004

Refrigerator: Zanussi, No.: ZLKI

- 262

Climatic chamberStudy code: 1 2 / 056

- 316AN Final Report Page 10 of 18

3.2. ANALYTICAL METHOD

3.2.1. HPLC Conditions:

Column: Zorbax Eclipse Plus C 8 1 50 × 4.6 mm, 5μm

Mobil e Phase : ACN : water

= 7 : 3

Flow: 1.0 ml/min.

Injection volume: 1 0 μl

Temperature: 25 °C

Detector: UV at 2 2 0 nm

Retention time: 4

. 6 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

- inject ion repeatability was tested with 7 consecutive injections of the 0. 2 and 1 0

μg/ml calibration standards. CV of the replicate injections was determined. The

repeatability was found to be 2

. 5 % or better.

Table 2.: The injection repeatability of the HPLC m ethod

Sample Measured parameter RSD (CV) %

Standard 0.2 μg/ml Retention time 0.0

Area 1.8

Standard 10 μg/ml Retention time 0.0

Area 2.5Study code: 1 2 / 056

- 316AN Final Report Page 11 of 18

3.3.3. Linearity

Linearity of the HPLC method was examined in 0. 1

- 2 0 μg/ml range. Eight

concentration leve ls were tested: 0. 1

, 0. 2

, 0. 5

, 1, 2, 5 , 10 and 2 0 μg/ml.

Five such series of standards were prepared from separate stock solutions and

analysed on five different days.

The detector response proved to be linear in 0. 1

– 2 0 μg/ml range. The calibration

grap h was calculated applying linear regression.

Deviation of the back

- calculated values was less 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 Param eters

Analytical occasion Intercept Slope Correlation Coefficient

1 6 October 201 2 687 22055 1.000

18 October 2012

- 305 23253 1.000

19 October 2012

- 197 21834 1.000

24 October 2012 368 21182 1.000

25 October 2012

- 435 22240 1.000

Figure 1: Lineari ty test on 19 October 201 2Study code: 1 2 / 056

- 316AN Final Report Page 12 of 18

3.3.4. Limit of Quantification

The lowest concentration of the calibration curve, 0. 1 μg/ml is considered to be the

quantification limit of the HPLC method.

3.3.5. Recovery, Accuracy and Precision

Recovery of test item wa s tested f ro m alga test solution s . Concentration s of the test

solutions were 1 and 2 0 mg/l.

Five replicate solutions were made.

Recovery is the measured value expressed in percentage of the nominal

concentration:

T M 100 **∗**

where:

M mean of me asurements

T nominal concentration

Precision is the standard deviation of replicates expressed as percentage of the

mean.



## ( **)**

M n M m

RSD n

i i

1

1 2

**− −** 

**=** 

## ∑

**=**

where:

RSD relative standard deviation

m i

- M individual deviations from the mean

n number of replicates

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

presented in Table 4.

Table 4: Recovery and Precision of the analytical method

Matrix Nominal

concentration Measured

concentration Recovery

% Precision

%

Alga test solution 1 mg/ l BQL

- -

20 mg/l 3.35 mg/l 15 22

BQL: below quantitation limit

The low recovery values are due to poor water solubility of the test item

.Study code: 1 2 / 056

- 316AN Final Report Page 13 of 18

3.3.6. Stability

Stock solution stability

Stock solution (1 mg/ml) prepared in water proved to be stable fo r at least 7 days at

5 **±** 3 °C. :

Peak area of the 1 0 μg/ml solution prepared from the 7 days old stock solution was

10 3 % of the peak area of the fresh one.

Stability of the samples in the autosampler

Calibration samples ( 0. 2 and 1 0 μg/ml) were reinjected after 20 hours storage

period in the autosampler. Peak areas after the storage period were 98 and 85 % of

the areas measured at the start. :

Stability of the test item in alga test solution

Test solutions were stored at room temperature. After one day fo rmulations were

analysed. :

Si n ce the test item is volatile and the test solutions were in an open vessel after one day

the concentration of the 20 μg/ml solution decreased below the quantitation level.

Presumably, this decrease was due to evaporation

, and n ot to decomposition of the

test item

.

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.Study code: 1 2 / 056

- 316AN Final Report Page 14 of 18

5. DISTRIBUTION OF THE FINAL REPORT

Sponsor:

- 1 × pdf

- file

Archives:

- 1 × original, bound

6. DEVIATION

The date of the Draft Report was 13 December 2012 instead of 1 6 Nove mber 2012,

because of the internal scheduling. This deviation does not have any effect on study

results.

There were a typo errors in the description of the test item. The correct molecular

formula is C 7 H 4 ClF 3 , and the correct molecular weight is 180.56.Study code: 1 2 / 056

- 316AN Final Report Page 15 of 18



## APPENDIXStudy code: 1 2 / 056

- 316AN Final Report Page 16 of 18



### 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 a bove GLP certificate is valid for this facility (now known as

CiToxLA B Hungary Ltd ) until the certificate expires (16 December 2012).Study code: 1 2 / 056

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- 316AN Final Report Page 18 of 18



### Certificate of Analysis