# Annex B



# Spirotetramat

B.5 Methods of analysis

Reviewed by PMRA, Canada

Peer – reviewed by: US-EPA, USA AGES, Austria– 2 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE OF CONTENTS

B.5.1 Analytical methods for technical active substance a nd formulation analysis

(Annex IIA 4.2, Annex IIIA 5.2) ....................

................................................... ........................3
B.5.1.1 Methods for the analysis of pure active substance i n the active substance as

manufactured (Annex II 4.2.1) ......................

................................................... .........................3 B.5.1.2 Applicability of existing CIPAC methods (Annex IIA 4.2.2) ............................................. .........4 B.5.1.3 Description of analytical methods for the determina tion of impurities (Annex IIA 4.2.3)..........5 B.5.1.4 Description of analytical methods for the determina tion of additive (Annex IIA 4.2.4).............5 B.5.1.5 Enforcement analytical methodology (Annex IIA 4.2.5 ).................................................. ..........5 B.5.1.6 Inter-Laboratory analytical methodology validaton ( Annex IIA 4.2.6)................................... ....5 B.5.1.7 Storage stability of working solutions in analytica l methodology (Annex IIA 4.2.6)..................5 B.5.1.8 Analytical methods for the determination of the act ive substance in plant protection

products (Annex IIIA 5.2) ..........................

................................................... .............................6 B.5.1.9 Applicability of existing CIPAC methods (Annex III A 5.2.3) ........................................... .......11 B.5.1.10 Analytical methods for the determination of relevan t impurities which are of

toxicological, ecotoxicological or environmetal con cern, in the preparation (Annex IIIA 5.2.4).............................................

................................................... .......................................11 B.5.1.11 Analytical methods for formulants or their constitu ents in the plant protection product.

(Annex IIIA 5.2.5).................................

................................................... ................................11

B.5.2 Analytical methods for the determination of residue s (all components included

in the residue definition proposed (Annex IIA 4.3, Annex IIIA 5.3)..................................12
B.5.2.1 Plant material.....................................

................................................... ..................................12 B.5.2.2 Foodstuff of animal origin, feeding stuffs.........

................................................... ....................24

B.5.3 Analytical methods for the determination of residue s in soil, water, sediment

and air (Annex IIA 4.4 to 4.7, Annex IIIA 5.4 to 5. 7) ................................................. ..........33
B.5.3.1 Soil (Annex IIA 4.4, Annex IIIA 5.4) ...............

................................................... ......................33 B.5.3.2 Water (Annex IIA 4.5, Annex IIIA 5.5)..............

................................................... ....................39 B.5.3.3 Sediment (Annex IIA 4.6, Annex IIIA 5.6)...........

................................................... .................44 B.5.3.4 Air (Annex IIA 4,7, Annex IIIA 5.7))...............

................................................... .......................44

B.5.4 Analytical methods for the determination or residue s in body fluids and tissues

(Annex IIA 4,8; Annex IIIA 5.8)....................

................................................... ......................47

B.5.5 Evaluation and assessment..........................

................................................... ....................48
B.5.5.1 Analytical methods for technical active substance a nd formulation analysis .........................48 B.5.5.2 Analytical methods for residues....................

................................................... .......................48

B.5.6 References relied on ...............................

................................................... ..........................51– 3 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.1 Analytical methods for technical active subst ance and formulation analysis (Annex IIA

4.2, Annex IIIA 5.2)

B.5.1.1 Methods for the analysis of pure active sub stance in the active substance as

manufactured (Annex II 4.2.1)

Report: KIIA 4.2.1/01, Hake, 2004 Title: Spirotetramat (BYI 08330) - Assay of Technic al Grade Active Ingredient -

HPLC - External Standard Report No & Document No AM001104MP1

M-084385-01-1 Dates of work: 2004-07-07 GLP Non GLP

Report: KIIA 4.2.1/02, Hake, 2004 Title: Validation of HPLC-method AM001104MP1 - BYI 08330 Assay of Technical Grade

Active Ingredient Report No & Document No VB1-AM001104MP1

M-084403-01-1 Dates of work: 2004-07-07 GLP Non GLP

The HPLC- method AM001104MP1 is used for the determ ination of spirotetramat in technical grade active substance.

Principle of the method : The components are separated by reverse phase high performance liquid chromatography (HPLC). The quantitative evaluation is carried out using the pe ak area of spirotetramat by comparison with the are a of the calibration substance according to the method of ex ternal standardization. The following table present s the details of method AM001104MP1.– 4 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Method ID AM001104MP1 Analyte Active Sample preparation Homogenize thoroughly approx. 5 g of the sample in an agate mortar. Transfer approx. 100 mg of this material accurately weighed to 0.00001 g to a 100 mL flask and make up to mark with acetonitril e. Instrument HPLC
Detector UV at 230 nm Column Phenomenex Luna C18, 200 mm × 4.6 mm, 3 μm Mobile phase Gradient of A and B where: A = 0.1% aqueous phosphoric acid B = methanol Time (min) % A % B Flow rate (mL/min)
0
36
39
41
45 45

45
5
5
45 55

55
95
95
55 1.2

1.2
1.2
1.2
1.2 Oven temperature 43 °C Quantitation External standard
Total run time 36 minutes Retention time Spirotetramat 31.0 minutes

The HPLC method AM001104MP1 for the determination o f spirotetramat in technical grade active substance has been completely validated by checking the param eters linearity, precision, accuracy, specificity a nd interference.

Linearity 6 concentrations with double measurements ; range 78 - 123 %; correlation coefficient r K : 0.9992; regression equation: y = 64.674x + 33.631. Chromatograms are given; the function is linear in the operating range.
Precision Repeatability 6 synthetic samples are measured; no outliers are reported, RSD: 0.50 %; acceptable according to the Horwitz equation. Accuracy 6 synthetic samples are measured; mean recovery: 100.3 %; RSD: 0.50 %. Specificity/Interference The UV-spectra of referenc e substance and analyte in the sample show no spectral difference; the retention times are ide ntical. No interferences are found.

Conclusion: The analytical method provided was fully validate d and assessed to be precise, selective and accurate for the determination of the active ingred ient spirotetramat.

B.5.1.2 Applicability of existing CIPAC methods (An nex IIA 4.2.2)

Up to now there is no CIPAC method available for the determination of spirotetramat in technical grade active substance.– 5 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.1.3 Description of analytical methods for the d etermination of impurities (Annex IIA 4.2.3)

Description in volume 4, confidential part

B.5.1.4 Description of analytical methods for the d etermination of additive (Annex IIA 4.2.4)

Refer to file of confidential information and data provided separately.

B.5.1.5 Enforcement analytical methodology (Annex I IA 4.2.5)

Not required for the methods for the analysis of th e active substance as manufactured.

B.5.1.6 Inter-Laboratory analytical methodology val idaton (Annex IIA 4.2.6)

Not required for the methods for the analysis of th e active substance as manufactured.

B.5.1.7 Storage stability of working solutions in a nalytical methodology (Annex IIA 4.2.6)

Not required for the methods for the analysis of th e active substance as manufactured.– 6 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.1.8 Analytical methods for the determination of the active substance in plant protection

products (Annex IIIA 5.2)

Analytical method for the determination of Spirotet ramat in Movento® 240 SC Insecticide

Report: KIIIA1 5.2/01, Zitzmann; 2004 Title: Determination of BYI 08330 in Formulations; Assay – HPLC – External Standard Report No & Document No AM002104MF1

M-068817-01-2 Dates of work 2004-05-11 GLP Non GLP

Report: KIIIA1 5.2/02, Odendahl; 2004 Title: Validation of HPLC-method AM002104MF1 -Deter mination of BYI 08330 in

FormulationsReport No & Document No VB1-AM002104MF1

M-068807-01-1 Dates of work 2004-05-05 GLP Non GLP

Report: KIIIA1 5.2/03, Odendahl; 2006 Title: Validation of HPLC-method AM002104MF1 -Deter mination of Spirotetramat in

FormulationsReport No & Document No VS3.1-AM002104MF1

M-269319-01-1 Dates of work 2006-04-07 GLP Non GLP

The HPLC method AM002104MF1 is applicable for the d etermination of the content of spirotetramat in formulations ( e.g . ‘Spirotetramat 240 g/L SC’).

Principle of the method : The components are separated by reversed phase chro matography using gradient elution (1L water + 5 ml 1N sulphuric acid / acetonitrile, 52/48 (v/v)). Aft er UV detection (240 nm), the quantitative evaluati on is carried out by comparing the peak areas with those of reference substances, using an external standard

. The following table presents the details of the met hod.– 7 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Method ID AM002104MF1

Sample preparation Transfer an amount of the homoge neous sample containing approx. 50 mg BYI 08330, accurately weighed to 0.1 mg, into a 100 mL volumetric flask. Add 5 mL of water to suspend the sample and then dissolve in ap prox. 50 mL acetonitrile. To dissolve the substance, place the flask in an ultra sonic bath for 15 min. Fill up the flask with water to just below the calibration mark and mix. Wait until the temperature has stabilized, then fill the flask up to the calibration mark with water.

Instrument HPLC

Detector UV at 240 nm

Column Prism RP 8, 5 mm * × 4.6 mm, 3 μm

Mobile phase Gradient of A and B where: A = 1 liter water + 5 mL 1N sulphuric acid B = acetonitrile

Time (min)

% A % B Flow rate (mL/min)

0
1.5
1.51
2.2
2.21
2.6 52

52
5
5
52
52 48

48
95
95
48
48 2

2
3
3
3
2

Oven temperature 50 °C

Quantitation External standard

Retention time 1.2 min

Total run time 3 minutes

The HPLC method AM002104MF1 has been completely val idated on the formulation ‘Spirotetramat 240 g/L SC (internal code: 0329757)’ by checking the parame ters linearity, precision, accuracy, specificity an d interference from excipients. Additionally interfer ence from excipients is checked on the formulation ‘Spirotetramat 240 g/L SC (internal code: 06424384) ’.– 8 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Linearity 6 concentrations with single measurements ; range 50 -150 %; correlation co-efficient r K : 1.0000; regression equation: y = 0.9968x + 0.3408. Chromatograms are given; the function is linear and proportional in the oper ating range. Precision 6 samples from one batch; single injectio n; no outliers; acceptable according to the Horwitz eq uation; relative standard deviation CV: 0.25 %. Accuracy 6 samples of laboratory-prepared synthetic formulation; recovery: 100.29 %, RSD: 0.48 %; the method shows no constant or proportional system atic error. Specificity The UV-spectra of active substance and reference substance show no spectral difference; the retention times of active substance and reference substance are identical.
Interference No interferences are found.

Conclusion : An analytical method was provided for the determina tion of the active. The method was assessed to be selective, precise and accurate for use as an enfor cement analytical method. However, the applicant mu st clarify what is the length of the HPLC column used.

The following deficiencies are identified and remai n to be addressed.

Title: Enforcement Analytical Method

Deficiencies: The length of the HPLC column used w as stated as 5 mm which is incorrect.

Clarification required: The applicant is requested to clarify the length of the HPLC column used.– 9 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Analytical method for the determination of Spirotet ramat in Movento® 150 OD Insecticide.

Report: KIIIA1 5.2/01, Zitzmann; 2004 Title: Determination of BYI 08330 in Formulations; Assay – HPLC – External Standard Report No & Document No AM002104MF1

M-068817-01-2 Dates of work 2004-05-11 GLP Non GLP

Report: KIIIA1 5.2/02, Odendahl; 2005 Title: Validation of HPLC-method AM002104MF1 -Deter mination of BYI 08330 in

FormulationsReport No & Document No VB2-AM002104MF1

M-260838-01-1 Dates of work 2005-11-14 GLP Non GLP

The HPLC method AM002104MF1 is applicable for the d etermination of the content of spirotetramat in formulations ( e.g . ‘Spirotetramat 150 g/L OD’).

Principle of the method : The components are separated by reversed phase chro matography using gradient elution (1L water + 5 ml 1N sulphuric acid / acetonitrile, 52/48 (v/v)). Aft er UV detection (240 nm), the quantitative evaluat ion is carried out by comparing the peak areas with those of reference substances, using an external standard

. The following table presents the details of the met hod.

Method ID AM002104MF1

Sample preparation Transfer an amount of the homoge neous sample containing approx. 50 mg BYI 08330, accurately weighed to 0.1 mg, into a 100 mL volumetric flask and dissolve in approx. 80 - 90 mL of solvent mixture of acetonitri le / tetrahydrofuran / 2-propanol, 2/3/5 and place the flask in an ultrasonic bath for 15 min. Fill up the flask with water to just below the calibration mark and mix. Wait u ntil the temperature has stabilized, then fill the flask up to the calibrati on mark with water.

Instrument HPLC

Detector UV at 240 nm

Column Prism RP 8, 5 mm * × 4.6 mm, 3 μm

Mobile phase Gradient A = 1 liter water + 5 mL 1N sulphuric acid Gradient B = acetonitrile

Time (min)

% A % B Flow rate (mL/min)

0
1.5
1.51
2.2
2.21
2.6 52

52
5
5
52
52 48

48
95
95
48
48 2

2
3
3
3
2

Oven temperature 50 °C– 10 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Quantitation External standard

Retention time 1.2 min

Total run time 3 minutes

The HPLC method AM002104MF1 has been completely validated on the formulation ‘Spirotetramat 150 g/L OD’ by checking the paramete rs linearity, precision, accuracy, specificity and interference from excipients.

Linearity 6 concentrations with single measurements ; range 50 -150 %; correlation co-efficient r K : 1.0000; regression equation: y = 0.9968x + 0.3408. Chromatograms are given; the function is linear and proportional in the oper ating range. Precision 6 samples from one batch; single injectio n; no outliers; acceptable according to the Horwitz eq uation; relative standard deviation CV: 0.50 %. Accuracy 6 samples of laboratory-prepared synthetic formulation; recovery: 100.29 %, RSD: 0.48 %; the method shows no constant or proportional system atic error. Specificity The UV-spectra of active substance and reference substance show no spectral difference; the retention times of active substance and reference substance are identical.
Interference No interferences are found.

Conclusion : An analytical method was provided for the determina tion of the active. The method was assessed to be selective, precise and accurate for use as an enfor cement analytical method. However, the applicant mu st clarify what is the length of the HPLC column used.– 11 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.1.9 Applicability of existing CIPAC methods (An nex IIIA 5.2.3)

A CIPAC method for the determination of spirotetram at in formulations is not available.

B.5.1.10 Analytical methods for the determination o f relevant impurities which are of

toxicological, ecotoxicological or environmental co ncern, in the preparation (Annex IIIA 5.2.4)

Neither Movento® 240 SC Insecticide nor Movento® 15 0 OD Insecticide contains any impurities which are of toxicological, ecotoxicological or environmental co ncern.

B.5.1.11 Analytical methods for formulants or their constituents in the plant protection product

(Annex IIIA 5.2.5)

With respect to toxicological, ecotoxicological or environmental aspects neither Movento® 240 SC Insecticide nor Movento® 150 OD Insecticide contain s formulants in relevant concentrations. Therefore a special analytical method and validation is not nee ded.– 12 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.2 Analytical methods for the determination of r esidues (all components included in the

residue definition proposed (Annex IIA 4.3, Annex I IIA 5.3)

B.5.2.1 Plant material

Report: KIIA 4.3/01, Schöning, R., Stuke, S., Billi an, P.; 2005 Title: Analytical method 00857 for the determinatio n of residues of BYI08330 (parent

compound and total residue), BYI08330-enol, BYI0833 0-ketohydroxy, BYI08330- mono-hydroxy and BYI08330-enol-Glc metabolite in/on plant material by HPLCMS/MS Report No &

Edition No MR-099/04 including Report MR-148/04: (Schoening, R , 2005) Supplement E001 of

the analytical method 00857 for the determination o f residues of BYI08330 (parent compound and total residue), BYI08330-enol, BYI0833 0-ketohydroxy, BYI08330- mono-hydroxy and BYI08330-enol-GLC metabolite in/on plant material by HPLCMS/MS M-253112-02-1

Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance document for generating and reporting M ethods of analysis SANCO/3029/99 US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 GLP yes

Report: KIIA 4.3/02, Freitag, T.; Daniels, M.; 2005 Title: Analytical Method 00857/M003 for the determi nation of residues of BYI08330 and

BYI08330-ketohydroxy metabolite in/on plant materia l by HPLC-MS/MS Report No & Edition No MR-098/05

M-263485-01-2

Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance document for generating and reporting M ethods of analysis SANCO/3029/99 US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 GLP yes

Report: KIIA 4.3/03, Spiegel, K.; 2006 Title: [Azaspirodecenyl-2- 14 C]BYI08330: Extraction efficiency testing of the re sidue

method for the determination of BYI08330, BYI08330- enol, BYI08330-ketohydroxy, BYI08330-monohydroxy and BYI08330-enol glucoside in cotton gin trash, in apple fruit and in lettuce using aged radioactive residue s Report No & Edition No MEF-05/022

M-248725-01-2

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340.

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes

Report: KIIA 4.3/04, Brookey, FM; 2006 Title: Independent laboratory validation of the res idue analytical method: “Analytical

method 00857 for the determination of residues of B YI08330 (parent compound and total residue of BYI08330), BYI08330-enol, BYI08330 -ketohydroxy, BYI08330- monohydroxy and BYI08330-enol-Glc metabolite in/on plant material by HPLCMS/MS” Report No & Edition No RAFNP008

M-277335-01-1– 13 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340.

EU-Guidelines SANCO/825/00 rev.7 Guidance Document on Residue Analytical Methods PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes

Report: KIIA 4.3/05, Pruitt WE; 2006 Title: FDA PAM Multiresidue Method (MRM) Report No & Edition No RAFNP007

M-277248-01-1 Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340 and OPPTTS

860.1360 Multiresidue Method Testing PMRA Regulatory Directive 98-02; DACO 7.2.4 GLP Yes

Report: KIIA 4.3/18, Schöning, R., Willmes, J., 2008 Title: Analytical method 01084 for the determinatio n of residues of spirotetramat

(BYI 08330), BYI08330-enol, BYI08330-ketohydroxy, B YI08330-mono-hydroxy and BYI08330-enol-glucoside metabolites in/on plant mat erial by HPLC-MS/MS Report No & Edition No MR-08/007

M-298287-01-1 Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance Document for generating and reporting m ethods of analysis SANCO/3029/99 US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes– 14 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance Document on residue analytical methods SANCO/3029/99 rev.7 of March 19, 2004 PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes

Report: KIIA 4.3/08, Klimmek, S.; 2005 Title: BYI08330-enol: examination of the applicabil ity of DFG method S 19 (extended and

revised revision) for the determination of residues of BYI08330-enol Report No & Edition No BAY-0519 Az. G05-0104

M-257512-01-1 Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance Document SANCO/825/00 BBA-guideline: Residue analytical methods for post- registration control purposes of July 21, 1998 GLP Yes

Report: KIIA 4.3/09, Freitag, T.; 2005 amended 02.0 6.2006 Title: Analytical method 00929 for the determinatio n of residues of BYI08330-

ketohydroxy-alcohol, BYI08330-desmethyl-ketohydroxy and BYI08330-desmethyldi-hydroxy in/on plant material by HPLC-MS/MS Report No & Edition No MR-026/05

M-256183-01-2 Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance Document for generating and reporting m ethods of analysis SANCO/3029/99 US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes

Report: KIIA 4.3/10, Klempner, A., Spiegel, K; 2006 Title: [Azaspirodecenyl-3- 14 C] BYI08330: extraction efficiency testing (radiova lidation) of

the residue method (00929) for the determination of BYI08330-ketohydroxy-alcohol, BYI08330-desmethyl-ketohydroxy and BYI08330-desmeth yl-di-hydroxy in plant samples using aged radioactive residues Report No & Edition No MEF-06/008

M-270054-02-2 Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340

PMRA Regulatory Directive 98-02; DACO 7.2.1, 7.2.2, 7.2.3– 15 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Principle of the Data-Gathering Method :

Analytical method 00857 Spirotetramat and the metabolites BYI 08330-enol, B YI 08330-ketohydroxy, BYI 08330-mono-hydroxy and BYI 08330-enol-Glc in/on plants were extracted usin g a blender with an acidic ACN/water mixture (4/1, v/v). After filtration and clean-up of the extract, the c orresponding stable isotopically labelled analytes were added as internal standards. Afterwards, the solution wa s made up to volume, diluted and subjected to HPLCMS/MS (TABLE B.5.2.1-1).

TABLE 5.2.1-1. Summary Parameters for the Analytical Method Used f or the Quantitation of Spirotetramat Residues in Plant Matrices. Method ID Analytical method 00857 Analyte(s) Spirotetramat, BYI08330-enol, BYI 08330- ketohydroxy, BYI 08330-mono-hydroxy and BYI 08330-enol-Glc Extraction solvent/technique acidic ACN/water mixtu re (4/1, v/v) Cleanup strategies filter through BondElut Clean po lyethylene frit Instrument/Detector Liquid chromatograph Agilent 11 00 with MS (API 4000 with ESI interface and MS detector) Column : Agilent Zorbax Eclipse-XDB-C8, 5 μ m, 150x4.6 mm i.d. with pre-column or equivalent. Mobile phase : Pump A: water/ACN (9/1, v/v), containing 0.22 mL/L formic acid. Pump B: ACN, containing 0.22 mL/L formic acid Pump C: water/ACN (1/1, v/v), containing 0.1 mL/L acetic acid or formic acid Ionization : ESI, positive
1 st MRM Spirotetramat 374 to 216 m/z BYI 08330-enol 302 to 216 m/z BYI 08330-ketohydroxy 318 to 268 m/z

BYI 08330-mono-hydroxy 304 to 254 m/z BYI 08330-enol-Glc 464 to 270 m/z
2 nd MRM* Spirotetramat 374 t o 302 m/z (confirmatory) BYI 08330-enol 302 to 270 m/z BYI 08330-ketohydroxy 318 to 214 m/z

BYI 08330-mono-hydroxy 304 to 119 m/z BYI 08330-enol-Glc 464 to 216 m/z Standardization method Mixed stable isotope interna l standards of spirotetramat, BYI 08330- enol, BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy and BYI 08330- enol-Glc. Stability of std solutions All analytes are stable in standard solutions for at least 4 weeks of storage in a refrigerator at 4°C ± 3°C under dark c onditions. Retention times Spirotetramat (~4.3 minutes), BYI 0 8330-enol (~2.7 minutes), BYI 08330-ketohydroxy (~3.2 minutes), BYI 08330-mono-hy droxy (~2.5 minutes) and BYI 08330-enol-Glc (~1.6 minutes).– 16 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.1-2 Characteristics for the Data-Gather ing Analytical Method 00857 used for the Quantitation of Spirotetramat Residues in Plant Mat rices. Analyte(s) Spirotetramat, BYI08330-enol, BYI 08330- ketohydroxy, BYI 08330-mono-hydroxy, and BYI 08330-enol-Glc Equipment ID Agilent 1100 HPLC system LOQ 0.01 ppm for each analyte in all plant matrices with the exception of hop cones. 0.1 ppm for each analyte in hop cones. LOD The LOD for all analytes was estimated to be 4- 10 fold lower than the corresponding LOQ. Accuracy/Precision Range of percent recoveries ± coefficient of variation indicated acceptable accuracy/precision in the spiking levels of 0.01 pp m and 1.0 ppm of each analyte for plant matrices (except hops), and 0.1-1 0 ppm in hop cones. Mean recoveries of all analytes from all tested mat rices were within the acceptable range of 70-120% with RSD <16%. Reliability of the Method/ [ILV] An independent laboratory method validation [ILV], Study No. RAFNP008, was conducted to verify the reliability of method N o. 00857 for the determination of spirotetramat residues in plant ma trices. The values obtained are indicative that method No. 00857 is re liable. Linearity The method/detector response was linear ( r 2 >0.997) within the range of 5 ng/L-50 μ g/L. Specificity The control chromatograms generally hav e no peaks above the chromatographic background and the spiked sample ch romatograms contain only the analyte peak of interest. Peaks w ere well defined and symmetrical. Two MRM transitions were monitored for each analyte/matrix for quantitation and confirmation purposes.

The extraction efficiency of method 00857 was teste d using cotton gin trash, lettuce, and apples conta ining aged radioactive residues from metabolism studies. The recoveries of the extracted radioactive residu es of spirotetramat and the metabolites in cotton gin tra sh, lettuce and apple samples using method 00857 we re in agreement with those generated in the metabolism st udies.

TABLE B.5.2.1-3. Extraction Efficiency of Method 00 857 using Radiolabelled Samples from Cotton, Lettuce and Apple Metabolism Studies. Crops Cotton gin trash (% extracted) Lettuce (% extracted) Apple (% extracted)
Method
00857 Metabolism

study Method

00857 Metabolis

m study Method

00857 Metabolism

study
% TRRs extracted 87.4 88.6 95.5 95.8 96.5 98.0 % of Compounds in the extract Spirotetramat 16.8 19.8 48.2 55.9 47.5 51.3
BYI 08330-enol 13.2 12.1 24.0 17.8 2.9 2.1
BYI 08330-enol-Glc 3.6 4.0 11.2 11.4 5.3 5.1
BYI 08330- ketohydroxy 31.8 29.7 12.0 6.2 8.7 7.7

BYI 08330-monohydroxy -- -- -- -- 15.6 15.6– 17 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Analytical method 00929 for data-gathering in rotat ional crops

Principle of the method : BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethyl- ketohydroxy and BYI 08330-desmethyl-di-hydroxy and the corresponding conjugates in/on plants were extracted using a blender with ACN/water (1/1, v/v)

. After filtration, the extract was concentrated to t he aqueous remainder and hydrolyzed with 3N hydroch loric acid, and cleaned up by eluting through the polysty renedivenylbenzene column. Afterwards, the corresponding stable isotopically labelled analytes were added as internal standards, the solution was made up to volume and subjected to HPLC-MS/MS (TABLE B.5 .2.1-4).

TABLE B.5.2.1-4. Summary Parameters for the Analytical Method Used f or the Quantitation of Spirotetramat Residues in Rotational Crop Plant Mat rices. Method ID Analytical method 00929 Analyte(s) BYI 08330-ketohydroxy-alcohol, BYI 08330 -desmethyl-ketohydroxy and BYI 08330-desmethyl-di-hydroxy Extraction solvent/technique ACN/water mixture (1/1 , v/v) Cleanup strategies filter through polystyrenediviny lbenzene column Instrument/Detector Liquid chromatograph Agilent 11 00 with MS (API 4000 with ESI interface and MS detector) Column : 1) Synergi Fusion-RP80, 125x4.6 mm; 5 μ m Phenomenex 2) Eclipse XDB-C8, 150x4.6 mm; 5 μ m Agilent Mobile phase : A: water/ACN (9/1, v/v), containing 0.1 mL/L aceti c acid. B: ACN, containing 0.1 mL/L acetic acid C: water/ACN (1/1, v/v), containing 0.1 mL/L aceti c acid Ionization : ESI, positive MRM of transitions: BYI 08330-ketohydroxy-alcohol 334 to 298 m/z BYI 08330-desmethyl-ketohydroxy 304 to 268 m/z BYI 08330-desmethyl-di-hydroxy 306 to 227 m/z

. Standardization method Mixed stable isotope interna l standards of BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethyl-ketohydroxy and BYI 08330-desme thyl-di-hydroxy. Stability of std solutions All analytes are stable in standard solutions for at least 2 months of storage in a refrigerator at 4 °C ± 3 °C under dark conditions.
Retention times Column 1 : BYI 08330-ketohydroxy-alcohol (~15.4 minutes), BYI 08330-desmethyl-ketohydroxy (~16.8 minutes), BYI 08330-desmethyl-di-hydroxy (~5.5 minutes). Column 2 : BYI 08330-ketohydroxy-alcohol (~12.1 minutes),

BYI 08330-desmethyl-ketohydroxy (~14.3 minutes), BYI 08330-desmethyl-di-hydroxy (~4.3 minutes).– 18 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.1-5. Characteristics for the Data-Gathe ring Analytical Method 00929 Used for the Quantitation of BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethylketohydroxy, and BYI 08330-desmethyl-di-hydroxy in Rotational Crop Plant Matrices. Analyte(s) BYI 08330-ketohydroxy-alcohol, BYI 08330 -desmethyl-ketohydroxy, and BYI 08330-desmethyl-di-hydroxy Equipment ID Agilent 1100 HPLC system LOQ 0.02 ppm for each analyte in all plant matrices

. LOD The LOD for all analytes was estimated to be at least 3-46% of the corresponding LOQ. Accuracy/Precision Range of percent recoveries ± coefficient of variation indicated acceptable accuracy/precision in the spiking levels of 0.02 ppm and 0.20 ppm of each analyte for plant matrices. Mean recoveries of all analytes from all tested matrices were within the a cceptable range of 70-120% with RSD <11%. Reliability of the Method/ [ILV] An ILV was not con ducted. Linearity The method/detector response was linear ( r ≥ 0.997) within the range of 0.02 – 20 μ g/L Specificity The control chromatograms generally hav e no peaks above the chromatographic background and the spiked sample chromatograms contain only the analyte peak of inte rest. Peaks were well defined and symmetrical. A second HPLC co lumn was used for confirmation purposes.

TABLE B.5.2.1-6. Extraction Efficiency of Method 00 929 using Radiolabelled Samples of Swiss Chard and Wheat Straw from Confined Rotationa l Crop Study. Crops Swiss Chard (% extracted) Wheat Straw (% extr acted)
Method
00929 Confined crop

rotation study Method 00929 Confined crop rotation study
% TRRs extracted 89.2 91.3 90.5 93.7 % of Compounds in the extract BYI 08330-desmethyl-di-hydroxy 6.8 7.7 22.2 25.5 BYI 08330-ketohydroxy-alcohol 5.0 5.8 13.8 13.4 BYI 08330-desmethyl-ketohydroxy 22.5 20.7 28.4 30.4

Enforcement Method 01084: During the evaluation the residue definition for pl ant matrices has changed. The notifier submitted an adequate analytical method which is evaluated by Au stria for EU.

Method 01084 is proposed as the enforcement method for the determination of residues of spirotetramat (BYI 08330), BYI08330-enol, BYI08330-ketohydroxy, B YI08330-mono-hydroxy and BYI08330-enolglucoside metabolites in/on plant material by HPLC- MS/MS.

Principle of the Method : Spirotetramat (BYI 08330) and its metabolites are e xtracted from potato (tuber), citrus (fruit), avoca do (fruit), tomato (fruit) and hop (cone dried) using a mixture of acetonitrile/water. After filtration the extrac ts, aliquots will be diluted with a mixture of acetonitrile/wate r. The residues are quantified by reversed phase HPLC with electrospray and MS/MS-detection (external calibration using standard in matrix).– 19 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.1-7. Summary Parameters for the Analytical Enforcement M ethod 01084 Used for the Quantitation of Spirotetramat, BYI08330-enol, B YI08330-ketohydroxy, BYI08330-mono-hydroxy, and BYI08330-enol-glucoside metabolites in Plant Matrices. Method ID Analytical method 01084 Analyte(s) Spirotetramat, BYI08330-enol, BYI08330-k etohydroxy, BYI08330-monohydroxy, and BYI08330-enol-glucoside metabolites in /on plant material Extraction solvent/technique Acidic ACN/water mixtu re (4/1, v/v, containing 0.22 mL formic acid/L) Cleanup strategies Celite 545 and filtration Instrument/Detector Liquid chromatograph e.g. Agile nt 1100 with Triple Quadrupole Tandem (MS/MS) e.g. API 4000 (AB MDS Sciex). Column : e.g.Zorbax, Eclipse XDB C8, 150 x 4.6 mm, 2.5μm, pre column

YMC ProC18, VS 12nm, S-3μm, 10 x 2.1 mm id Mobile phase : Mobile Phase A: Water/acetonitrile (9/1, v/v) + 10 mmol/L ammonium formate + 220 μL formic acid/L

Mobile Phase B: Acetonitrile + 10 mmol/L ammonium f ormate + 220 μL formic acid/L Isocratic Pump: Acetonitrile/Water (1/1, v/v) + 10 mmol/L ammonium formate + 220 μL formic acid/L Different gradients are used for the determination of Spirotetramat, BYI08330-enol, BYI08330-ketohydroxy, and BYI08330-m onohydroxy and the determination of BYI08330-enol-gluc oside Ionization: ESI, positive

MRM of transitions (the second MRM was taken for co nfirmation): BYI08330 m/z 374 → 216 and m/z 374 → 302 BYI08330-enol m/z 302 → 216 and m/z 302 → 270 BYI08330-ketohydroxy m/z 318 → 268 and m/z 318 → 214 BYI08330-monohydroxy m/z 304 → 254 and m/z 304 → 119 BYI08330-enol-glucoside m/z 464 → 270 and m/z 464 → 216 Standardization method Linear regression with 1/x w eighting Stability of std solutions All analytes are stable in standard solutions up to 4 weeks of storage in a refrigerator at 4°C ± 3°C under dark conditions. Retention times BYI 08330 approx. 4.7 min BYI08330-enol approx. 2.1 min BYI08330-ketohydroxy approx. 2.4 min BYI08330-mono-hydroxy approx. 2.0 min BYI08330-enol-glucoside approx. 1.7 min

TABLE B.5.2.1-8. Characteristics for the Enforcemen t Analytical Method 01084 Used for the Quantitation of Spirotetramat Residues in Plant Mat rices. Analyte(s) Spirotetramat, BYI08330-enol, BYI08330-k etohydroxy, BYI08330-monohydroxy, and BYI08330-enol-glucoside metabolites in /on plant material LOQ 0.01 mg/kg (expressed as parent equivalents) in avocado (fruit), orange (fruit), potato (tuber), and tomato (fruit) 0.1 mg/kg in hop cones LOD 0.01 mg/kg in hop cones, 0.001 mg/kg in all oth er crops tested.– 20 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Accuracy/Precision Mean recoveries for each fortifi cation level and analyte are between 70- 110 % for all matrices. Relative standard deviations were below 20 % for al l analytes and sample materials. Detailed recovery information is reported below. Reliability of the Method/ [ILV] An ILV has not yet been performed on this method. Linearity The correlation between the injected amou nt of substance and the detector response was linear for solvent standards and matrix matched standards ranging from 0.025 to 10 μg/L. The correl ation coefficients were between 0.9876 (lowest coefficient for BYl08330-eno l-glucoside in solvent and was only for demonstration of matrix effects) a nd 0.9999 for all items. For quantitation of the recoveries only matrix matc hed standards were relevant. Representative linearity plots for matrix citrus are provided for each analyte (n = 5). Specificity Residues in control samples were below 30 % of the respective LOQ levels. The recoveries were not corrected for inter ferences. LC-MS/MS as a detection technique can be considered to be highl y specific and therefore no confirmatory technique is required. Two MRM transitions were monitored for each analyte /matrix for quantitation and confirmation purposes. Chromatogra ms of control samples are available

TABLE B.5.2.1-9. Validation data for analytical me thods for the determination of residues of the active substance in food of plant origin. Analyte (each metabolite is expressed as parent equivalent) Matrix Fortification Level [mg/kg] n Mean

Recovery [%] RSD

[%] Overall Mean

Recovery [%] Overall RSD

[%]

0.10 5 90 1.5 Hop Cone Dried 1.0 5 79 1.9 85 7.0

0.01 5 94 2.6 Avocado Fruit 0.10 5 93 3.8 94 3.2

0.01 5 93 2.6 Orange Fruit 0.10 5 91 0.6 92 2.2

0.01 5 93 1.3 Potato Tuber 0.10 5 89 1.8 91 2.7

0.01 5 92 1.5 BYI 08330 (1 st MRM)

Tomato

Fruit 0.10 5 94 0.6 93 1.4

0.10 5 92 3.2 Hop Cone Dried 1.0 5 80 1.9 86 8.1

0.01 5 93 2.5 Avocado Fruit 0.10 5 94 2.3 94 2.3

0.01 5 90 2.9 Orange Fruit 0.10 5 91 1.4 91 2.2

0.01 5 92 2.1 Potato Tuber 0.10 5 87 1.1 90 3.5 BYI 08330 (2 nd MRM)

Tomato 0.01 5 93 1.8 93 1.4– 21 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Analyte (each metabolite is expressed as parent equivalent) Matrix Fortification Level [mg/kg] n Mean

Recovery [%] RSD

[%] Overall Mean

Recovery [%] Overall RSD

[%]

Fruit 0.10 5 93 1.0

0.10 5 100 1.8 Hop Cone Dried 1.0 5 72 2.5 86 17.2

0.01 5 93 3.2 Avocado Fruit 0.10 5 88 1.0 90 3.8

0.01 5 87 1.4 Orange Fruit 0.10 5 89 1.2 88 1.6

0.01 5 88 1.2 Potato Tuber 0.10 5 90 0.9 89 1.6

0.01 5 89 1.0 BYI08330- enol (1 st MRM)

Tomato

Fruit 0.10 5 92 0.6 91 1.9

0.10 5 102 1.9 Hop Cone Dried 1.0 5 71 2.3 87 18.9

0.01 5 95 1.5 Avocado Fruit 0.10 5 88 1.0 91 4.3

0.01 5 86 2.1 Orange Fruit 0.10 5 88 1.3 87 2.4

0.01 5 88 1.9 Potato Tuber 0.10 5 90 0.8 89 1.7

0.01 5 89 1.8 BYI08330- enol (2 nd MRM)

Tomato

Fruit 0.10 5 91 0.6 90 1.9

0.10 5 97 5.0 Hop Cone Dried 1.0 5 72 2.1 85 15.8

0.01 5 94 4.7 Avocado Fruit 0.10 5 87 1.0 91 5.2

0.01 5 95 3.0 Orange Fruit 0.10 5 89 2.2 92 4.1

0.01 5 96 5.1 Potato Tuber 0.10 5 96 6.0 96 5.3

0.01 5 91 3.4 BYI08330- ketohydroxy (1 st MRM

Tomato

Fruit 0.10 5 94 2.1 92 3.1

0.10 5 96 3.6 Hop Cone Dried 1.0 5 72 2.9 84 15.9

0.01 5 91 2.5 Avocado Fruit 0.10 5 91 2.1 91 2.2

0.01 5 92 2.2 Orange Fruit 0.10 5 91 1.7 92 2.0

0.01 5 94 4.2 Potato Tuber 0.10 5 99 6.9 96 6.1

0.01 5 96 4.2 BYI08330- ketohydroxy (2 nd MRM

Tomato

Fruit 0.10 5 95 1.7 96 3.1

0.10 5 100 3.3 Hop Cone Dried 1.0 5 72 3.2 86 17.2 BYI08330-

monohydroxy Avocado 0.01 5 97 2.1 92 5.3– 22 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Analyte (each metabolite is expressed as parent equivalent) Matrix Fortification Level [mg/kg] n Mean

Recovery [%] RSD

[%] Overall Mean

Recovery [%] Overall RSD

[%]

Fruit 0.10 5 88 0.5

0.01 5 87 3.6 Orange Fruit 0.10 5 90 1.0 88 2.8

0.01 5 93 2.0 Potato Tuber 0.10 5 94 1.7 93 2.0

0.01 5 93 1.7 (1 st MRM)

Tomato

Fruit 0.10 5 89 0.8 91 2.6

0.10 5 97 2.8 Hop Cone Dried 1.0 5 74 3.4 85 14.4

0.01 5 98 3.6 Avocado Fruit 0.10 5 91 1.0 95 4.4

0.01 5 87 3.5 Orange Fruit 0.10 5 90 1.0 89 3.3

0.01 5 89 4.9 Potato Tuber 0.10 5 104 1.5 96 8.9

0.01 5 95 2.1 BYI08330-

monohydroxy (2 nd MRM)

Tomato

Fruit 0.10 5 91 2.0 93 3.1

0.10 5 106 6.2 Hop Cone Dried 1.0 5 85 4.8 96 12.6

0.01 5 101 6.7 Avocado Fruit 0.10 5 107 2.1 104 5.6

0.01 5 88 5.2 Orange Fruit 0.10 5 98 2.0 93 7.1

0.01 5 90 5.5 Potato Tuber 0.10 5 97 2.7 93 5.4

0.01 5 88 5.5 BYI08330- Glucoside (1 st MRM)

Tomato

Fruit 0.10 5 88 2.2 88 4.0

0.10 5 100 4.1 Hop Cone Dried 1.0 5 80 6.9 90 13.0

0.01 5 101 6.8 Avocado Fruit 0.10 5 103 3.1 102 5.0

0.01 5 84 4.4 Orange Fruit 0.10 5 94 4.5 89 7.4

0.01 5 94 3.7 Potato Tuber 0.10 5 99 5.5 97 5.3

0.01 5 85 3.0 BYI08330- Glucoside (2 nd MRM)

Tomato

Fruit 0.10 5 92 4.9 89 5.6

Conclusion: The analytical method 01084 is sufficiently validat ed to monitor the active substance and the metaboli tes according to the current residue definition in plan t material. The calibration range for hop should be up to 12 μg /L to fulfill the requirement that the upper calibr ation value should exceed at least 20% the highest measured lev el. The given value of10 μg/L can be accepted since appropriate dilution can be carried out to be withi n the calibration range.– 23 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

An independent laboratory validation is not yet ava ilable. Multiresidue Method

Principle of the Method : Residues of spirotetramat, BYI 08330-enol, BYI 0833 0-ketohydroxy, BYI 08330-mono-hydroxy, BYI 08330- enol-Glc, and BYI 08330-enol-GA were subjected to t he appropriate steps of the multiresidue methodolog y described in the United States Food and Drug Admini stration (FDA) Pesticide Analytical Manual (PAM), Volume 1. The pertinent protocols tested are in TAB LE B.5.2.1-9.

TABLE B.5.2.1-10. Results of Multiresidue Methods Testing with Spirotetramat, BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330-mono-hydroxy, BYI 08330-enol-Glc, and BYI 08330-enol-GA.
PAM 1 Protocols Results Comments A BYI 08330-mono-hydroxy was

found to be naturally fluorescent. BYI 08330-mono-hydroxy is a minor metabolite and not being proposed as an enforcement target compound; therefore, no further testing was performed. B Not performed Test substances are not acids or ph enols. C Ucceptable Nitrogen-specific modules DG5 and DG17 were unsuccessful at chromatographing the test substances due to no peak or the response wasoutside the relative retention time window. Nonspecific modules DG1, DG13 and DG18 were unsuccessful at chromatographing the test substances due to no peak, multiple peaks, or the response was outside the relative retention time window. D, E, F Not performed No further analyses were perf ormed for Protocols D, E and F due to unsuccessful results with Protocol C

. G Not performed Test substances are not substituted ureas.

Conclusion: Analytical method 00857 quantitating spirotetramat, BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330- mono-hydroxy and BYI 08330-enol-Glc was adequately validated on a wide variety of plant commodities an d is suitable for data-gathering and enforcement purp oses. Analytical method 00929 quantitating residue s of BYI 08330-ketohydroxy-alcohol, BYI 08330-desmethyl- ketohydroxy, and BYI 08330-desmethyl-di-hydroxy in rotational crops was adequately validated on wheat (grain, forage, straw and flour), cotton undelinted seeds, Swiss chard, peanut (oil and nutmeat), and sugar be ets (roots and molasses). The extraction efficienci es of methods 00857 and 00929 were tested using samples c ontaining aged radioactive residues from metabolism studies. The recoveries of the extracte d radioactive residues of spirotetramat and the metabolites were in agreement with those generated in the metabolism studies.

The test substances spirotetramat, and metabolites BYI 08330-enol, BYI 08330-ketohydroxy, BYI 08330- enol-Glc, and BYI 08330-enol-GA could not be determ ined by the FDA PAM Volume 1 multiresidue methods.– 24 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.2.2 Foodstuff of animal origin, feeding stuffs

Report: KIIA 4.3/11, Freitag, Th., Wolters, A.; 2006 Title: Analytical method 00966 for the determinatio n of residues of BYI 08330, BYI08330-

enol, and BYI08330-enol-GA in/on matrices of animal origin by HPLC-MS/MS Report No & Edition No MR-150/05

M-266199-01-2 Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance Document for generating and reporting m ethods of analysis SANCO/3029/99 US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes

Report: KIIA 4.3/12, Duah, F.; 2006 Title: Request for waiver of the requirement for ra diovalidation of analytical method for the

determination of BYI08330 residues in animal matric es Report No & Edition No RAFNX039

M-277087-01-1 Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340, Residue

Analytical Method-Extraction Efficiency PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes

Report: KIIA 4.3/13, Perez R; 2006 Title: Independent method validation of Bayer metho d no. 00966 (report no. MR-150/05)

BYI08330: Analytical method 00966 for determination of residues of BYI08330, BYI08330-enol and BYI08330-enol-glucuronide in/on m atrices of animal origin by HPLC-MS/MS Report No & Edition No RAFNP009

M-277298-01-1 Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340

PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes

Report: KIIA 4.3/14, Coopersmith H; 2006 Title: Analytical method for the determination of r esidues of BYI08330, BYI08330-enol

and BYI08330-enol-GA in/on matrices of animal origi n by HPLC-MS/MS (modification of analytical method 00966) Report No & Edition No RAFNP017

M-277359-02-1 Guidelines: US-EPA Residue Chemistry Test Guideline OPPTS 860.1340

PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 EU Council Directive 91/414/EEC amended by Commissi on Directive 96/68/EC EU Guidance Document for generating and reporting m ethods of analysis SANCO/3029/99 GLP Yes

Report: KIIA 4.3/15 Freitag, Th., Wolters, A.; 2006 Title: Analytical method 00969 for determination of residues of BYI08330-enol in/on

matrices of animal origin by HPLC-MS/MS Report No & Edition No MR-160/05

M-265407-01-1 Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance Document for generating and reporting m ethods of analysis SANCO/3029/99 US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2 , 7.2.3 GLP Yes– 25 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Report: KIIA 4.3/16 Bacher, R.; 2006 Title: Independent laboratory validation of BCS Ana lytical Method 00969 for the

determination of residues of BYI08330-enol in mater ials of animal origin Report No & Edition No P/B 964 G

M-269571-01-1 Guidelines: EU Council Directive 91/414/EEC amended by Commission Directive 96/68/EC

EU Guidance document for generating and reporting M ethods of analysis SANCO/3029/99 US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 PMRA Regulatory Directive 98-02: DACO 7.2.1, 7.2.2, 7.2.3 GLP Yes– 26 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Data-Gathering Method 00966

Principle of the Method: Spirotetramat and the metabolites BYI 08330-enol an d BYI 08330-enol-GA were extracted from the sample material (fat, liver, kidney) using a blender with an acidic acetonitrile/water mixture (7/3, v/v). M ilk and muscle were extracted using a blender with acidic a cetonitrile. After subsequent clean-up by exhausti ng the extract through a Varian MEGA BE-C18 cartridge, the corresponding stable isotopically labelled analyte s were added as internal standards. Afterwards, the solution was made up to volume, diluted and subject ed to HPLC-MS/MS.

Bayer Method FN-006-A06-01 Analytical method 00966 was modified after an ILV. As recommended by the ILV laboratory, the method was modified (Bayer Method FN-006-A06-01) to provid e an option to remove a cleanup step for livestock tissues; while milk, milk fat and whey samples are purified by C18 solid-phase extraction (SPE) prior to HPLC-MS/MS analysis.

TABLE B.5.2.2-1. Summary Parameters for the Analytical Method Used f or the Quantitation of Spirotetramat Residues in Livestock Matrices. Method ID Analytical Method 00966 Bayer Method FN- 006-A06-01 (modification of Analytical Method 00966) Analyte(s) Spirotetramat, BYI 08330-enol, BYI 08330 –enol-GA
Extraction solvent/technique Fat, liver & kidney - homogenize with acidic aceton itrile/water (7/3, v/v); Milk & muscle - homogenize with acidic acetonitrile. Cleanup strategies By filtering through a Varian ME GA BEC18 cartridge. After homogenization, livestock tissues were stirred well and allowed to separate until solids were at the bottom of the jar). Milk, milk fat or whey samples were cleaned up by filtering through a Varian MEGA BE-C18 cartridge.– 27 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.2-1. Summary Parameters for the Analytical Method Used f or the Quantitation of Spirotetramat Residues in Livestock Matrices. Instrument/Detector Liquid chromatograph Agilent 11 00 with mass spectrometer (API 4000 with ESI interface and mass spectrometric detector). Column : Agilent Zorbax EclipseXDB-C8, 5 μ m, 150x4.6 mm i.d. with pre-column or equivalent. Mobile phase : Pump A: water/acetonitrile (9/1, v/v), containing 0.22 mL/L formic acid. Pump B: acetonitrile, containing 0.22 mL/L formic acid. Pump C: water/acetonitrile (1/1, v/v), containing 0.1 mL/L acetic acid or formic acid.

Ionization : ESI, positive
1 st MRM Spirotetramat: 374 to 216 m/z BYI 08330-enol: 302 to 216 m/z BYI 08330-enol-GA: 478 to 302 m/z
2 nd MRM Spirotetramat: 374 to 302 m/z BYI 08330-enol: 302 to 270 m/z BYI 08330-enol-GA: 478 to 216 m/z Thermo Finnigan Surveyor instrument.

Column : Gemini C6-Phenyl, 3

μ m, 50x2.0 mm i.d. with precartridge Serial Nos. 333946-3 &
347941-1.

Mobile phase : A: water/acetonitrile (9/1, v/v), containing 0.22 mL/L formic acid. B: acetonitrile, containing 0.22 mL/L formic acid.

Scanning mode : SelectedReaction Monitoring (SRM), negative ion mode. Parent ion Daughter ion Spirotetramat 374 to 216 BYI 08330-enol 302 to 216 BYI 08330-enol-GA 478 to 302

Standardization
method Mixed stable isotope internal standards of Spirotet ramat, BYI 08330-enol and BYI 08330-enol-GA Stability of std solutions Spirotetramat, BYI 08330-enol, BYI 08330-ketohydrox y, BYI 08330-mono-hydroxy and BYI 08330-enol-GA are stable in standard soluti ons for at least four weeks of storage in a refrigerator at 4oC ± 3oC under dark c onditions. Retention times Spirotetramat (~4.6 minutes), BYI 08330-enol (~3.9 minutes), BYI 08330- enol-GA (~1.9 minutes) Not Reported– 28 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.2-2. Characteristics for the Data-Gathe ring Analytical Method Used for the Quantitation of Spirotetramat Residues in Livestock Matrices. Analyte(s) Spirotetramat, BYI 08330-enol, BYI 0833 0-enol-GA Equipment ID Agilent 1100 HPLC system LOQ 0.01 ppm for each analyte in all livestock matr ices, except milk. 0.005 ppm for each analyte in milk. LOD The LOD for all analytes was estimated to be 10 fold lower than the corresponding LOQ. Accuracy/Precision Range of percent recoveries ± co efficient of variation indicated acceptable accuracy/precision in the spiking levels of 0.01 pp m and 0.10 ppm for muscle (poultry & cattle), cattle kidney, liver & f at; and 0.005 ppm and 0.050 ppm for milk. The percent recoveries from al l matrices were 68- 101% for spirotetramat, 75-102% for BYI 08330-enol, and 69-105% for BYI 08330-enol-GA. Average recoveries ranged from 72% to 99% and relative standard deviations were <10%. Reliability of the Method/ [ILV] An ILV was conducted to verify the reliability of m ethod No. 00966 for the determination of spirotetramat, BYI 08330-enol and BYI 08330-enol-GA residues in livestock matrices. The values obtaine d are indicative that method No. 00966 is reliable. Linearity The method/detector response was linear ( coefficient of determination, r 2 = 0.999) within the range of 0.02-20 ug/L. Specificity The control chromatograms generally hav e no peaks above the chromatographic background and the spiked sample ch romatograms contain only the analyte peak of interest. Peaks w ere well defined and symmetrical. Two MRM transitions were monitored for each matrix and each analyte for quantitation and confirmation purp oses.

Independent Laboratory Validation

Analytical method 00966

An ILV was conducted by ADPEN Laboratories, Inc., J acksonville, FL. Homogenized beef liver control samples were spiked with spirotetramat, BYI 08330-e nol and BYI 08330-enol-GA at 0.01 ppm (LOQ), 0.02 ppm and 0.10 ppm, and analyzed by HPLC-MS/MS accord ing to method 00966. During the first trial, the method was followed as written. The recoveries of spirotetramat in beef liver were low at all spiking levels while the recoveries of BYI 08330-enol were high. The method was modified in that the extraction solv ent was added within 5 minutes of spiking and each samp le was extracted within 30 minutes. As well, the S PE cleanup step with the Varian MEGA BE-C18 was remove d. The second ILV trial using the modified method was successful and supports the method LOQ of 0.01 ppm. At the second trial, individual recoveries of spirotetramat, BYI 08330-enol, and BYI 08330-enol-G A from spiked beef liver ranged from 72% to 99%. Average recoveries ranged from 76% to 96% with RSD ≤ 5%.– 29 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.2-3. Recovery Results Obtained by an In dependent Laboratory Validation of the Analytical Method 00966 for the Determination of sp irotetramat Residues in Livestock Matrices. Matrix Spiking Level (ppm) Recoveries Obtained (%) Mean Recovery ± SD (CV) (%) Spirotetramat Beef liver 0.01 93, 85, 93, 91, 90 90 ± 3 (4) 0.02 93, 92, 93, 89, 96 93 ± 3 (3) 0.10 86, 90, 94, 96, 95 92 ± 4 (4)
BYI 08330-enol 0.01 99, 96, 94, 88, 88 93 ± 5 (5) 0.02 91, 98, 99, 97, 93 96 ± 3 (3) Beef liver 0.10 91, 93, 97, 93, 98 94 ± 3 (3)
BYI 08330-enol-GA 0.01 80, 76, 79, 72, 76 76 ± 3 (4) 0.02 76, 77, 77, 79, 76 77 ± 2 (1) Beef liver 0.10 72, 79, 76, 76, 82 77 ± 5 (4)

Conclusion : Analytical method 00966 (updated to FN-006-A06-01) quantitating spirotetramat, BYI 08330-enol and BYI 08330-enol-GA was adequately validated and is suita ble for data-gathering purposes in cattle and poult ry tissues, milk and eggs. Although the proposed enfo rcement method 00969 measures only metabolite BYI 08330-enol in livestock matrices, it was successful ly validated in cattle milk, muscle and poultry egg s. A waiver was submitted by the applicant to remit the requirement for an extraction efficiency study. An extraction efficiency study was not considered nece ssary since method 00966 uses the same extraction solvents as those used in the livestock metabolism study.– 30 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Enforcement method 00969:

Principle of the method: BYI 08330-enol was extracted from egg and kidney sa mples using a blender with an acetonitrile/water mixture (7/3, v/v). Milk and muscle samples were e xtracted using a blender with acetonitrile. After subsequent clean-up of the extract through a Varian MEGA BE-C18 cartridge, the solution was made up to volume, diluted and subjected to HPLC-MS/MS.

TABLE B.5.2.2-4. Summary Parameters for the Analytical Enforcement M ethod Used for the Quantitation of BYI 08330-enol Residues in Livestoc k Matrices. Method ID Analytical method 00969 Analyte(s) BYI 08330-enol Extraction solvent/technique Kidney & egg - homogen ize with acetonitrile/water (7/3, v/v) Milk & muscle - homogenize with acetonitrile Cleanup strategies By filtering through a Varian ME GA BE-C18 cartridge.
Instrument/Detector same as described for method 00 966 Standardization method External standards; matrix-m atched standards Stability of std solutions Spirotetramat-enol is st able in standard solutions for at least four weeks of storage in a refrigerator at 4oC ± 3oC under dark c onditions. Retention times BYI 08330-enol (~3.8 minutes)

TABLE B.5.2.2-5. Characteristics for the Enforcemen t Analytical Method 00969 Used for the Quantitation of BYI 08330-enol Residues in Livestoc k Matrices. Analyte(s) BYI 08330-enol Equipment ID Agilent 1100 HPLC system LOQ 0.01 ppm (kidney, muscle, egg) 0.005 ppm (milk) LOD 0.001 ppm (kidney, muscle, egg) 0.0005 ppm (milk) Accuracy/Precision Range of percent recoveries ± co efficient of variation ( ≤ 6%) indicating acceptable accuracy/precision in the range of spiki ng levels of 0.01 ppm and 0.10 ppm for cattle kidney & muscle, and egg; 0 .005 ppm and 0.05 ppm for milk. Reliability of the Method/ [ILV] An ILV was conduct ed to verify the reliability of method No. 00969 fo r the determination of BYI 08330-enol residues in livesto ck matrices. The values obtained are indicative that method No. 0096 9 is reliable. The linearity range of ILV is 0.050 μg/L to 5.0 μg/L wh ich is different from the range of 0.02 ng/mL to 20 μg/L reported for the pr imary method. Linearity The method/detector response was linear ( r= 0.999) within the range of 0.02-20 ug/L. Specificity The control chromatograms generally hav e no peaks above the chromatographic background and the spiked sample ch romatograms contain only the analyte peak of interest. Peaks w ere well defined and symmetrical.– 31 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.2-6. Recovery Results from Method Valid ation of Livestock Matrices using the Enforcement Analytical Method 00969. Standards Wer e Prepared in Acetonitrile/Water (15/85, v/v containing 0.22 mL/L formic acid). Matrix Spiking Level (mg/kg) Recoveries Obtained Mean Recovery ± SD (CV)

BYI 08330-enol 0.01 95, 98, 93, 92, 88 93 ± 4 (4) Cattle muscle 0.10 90, 84, 94, 95, 100 93 ± 6 (6) 0.01 88, 95, 92, 91, 90 91 ± 3 (3) Cattle kidney 0.10 94, 95, 94, 95, 94 94 ± 1 (1) 0.005 99, 99, 96, 99, 103 99 ± 2 (3) Cattle milk 0.050 97, 97, 94, 95, 97 96 ± 1 (1) 0.01 95, 88, 94, 91, 91 92 ± 3 (3) Poultry egg 0.10 90, 94, 94, 94, 94 93 ± 2 (2)

TABLE B.5.2.2-7. Characteristics for the Enforcemen t Analytical Method 00969 Used for the Quantitation of BYI 08330-enol Residues in Livestoc k Matrices. Analyte(s) BYI 08330-enol Equipment ID Agilent 1100 HPLC system LOQ 0.01 ppm (kidney, muscle, egg) 0.005 ppm (milk) LOD 0.001 ppm (kidney, muscle, egg) 0.0005 ppm (milk) Accuracy/Precision Range of percent recoveries ± co efficient of variation ( ≤ 6%) indicating acceptable accuracy/precision in the range of spiki ng levels of 0.01 ppm and 0.10 ppm for cattle kidney & muscle, and egg; 0 .005 ppm and 0.05 ppm for milk. Reliability of the Method/ [ILV] An ILV was conduct ed to verify the reliability of method No. 00969 fo r the determination of BYI 08330-enol residues in livesto ck matrices. The values obtained are indicative that method No. 0096 9 is reliable. The linearity range of ILV is 0.050 μg/L to 5.0 μg/L wh ich is different from the range of 0.02 ng/mL to 20 μg/L reported for the pr imary method. Linearity The method/detector response was linear ( r= 0.999) within the range of 0.02-20 ug/L. Specificity The control chromatograms generally hav e no peaks above the chromatographic background and the spiked sample ch romatograms contain only the analyte peak of interest. Peaks w ere well defined and symmetrical.

Independent Laboratory Validation

Analytical method 00969

An ILV for analytical method 00969 was conducted by PTRL Europe, Germany; using bovine meat, milk and poultry egg as the sample matrices. Control sample s of meat and egg were spiked with BYI 08330-enol a t 0.01 and 0.10 ppm, and milk samples were spiked at 0.005 and 0.05 ppm. Due to significant modificatio ns in the HPLC conditions, the first trial was conside red unsuccessful. After consultation with the spon sor, the second trial was successful using HPLC gradient con ditions similar to those given in the original meth od. The mean recoveries were 95-99% with RSD ≤ 8%.– 32 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.2.2-8. Recovery Results Obtained by an IL V of the Analytical Method 00969 for the Determination of BYI 08330-enol in Livestock Matric es Matrix Spiking Level (mg/kg) Recoveries Obtained Mean Recovery  SD (CV)

BYI 08330-enol 0.005 93, 98, 94, 103, 103 98 ± 5 (5) Cattle milk 0.050 99, 99, 99, 100, 93 98 ± 3 (3) 0.01 106, 90, 88, 98, 100 97 ± 8 (8) Cattle muscle 0.10 96, 96, 96, 96, 101 97 ± 2 (2) 0.01 95, 91, 100, 96, 93 95 ± 4 (4) Poultry egg 0.10 96, 100, 98, 102, 98 99 ± 2 (2)

CONCLUSION

Although the proposed enforcement method 00969 meas ures only metabolite BYI 08330-enol in livestock matrices, it was successfully validated in cattle m ilk, muscle and poultry eggs.

Remark by EU : According to Section Residue only the metabolite BYI08330-enol is expected in animal matrices therefore the current residue definition f or monitoring (i.e.BYI08330 and BYI08330-enol) shou ld be changed.– 33 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.3 Analytical methods for the determination of r esidues in soil, water, sediment and air

(Annex IIA 4.4 to 4.7, Annex IIIA 5.4 to 5.7)

B.5.3.1 Soil (Annex IIA 4.4, Annex IIIA 5.4)

Report: KIIA 4.4/01, Brumhard, B. 2006 (MR-06/034) Title: Analytical Method 00986 for the Determinatio n of Residues of BYI08330 and

BYI08330-enol in Soil by HPLC-MS/MS Report No & Edition No MR-06/034 (Method No. 00986)

M-268586-01-1 Guidelines: EC Guidance Document on Residue Analyti cal Methods, SANCO/825/00 rev.7 of

March 17, 2004. BBA Guideline: Residue Analytical M ethods for Post-Registration Control Purposes of July 21, 1998. Commission Direc tive 96/46/EC amending Council Directive 91/414/EEC of 16 July 1996. US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 GLP Fully GLP compliant - laboratory certified by G erman “Ministerium für Umwelt,

Raumordnung und Landwirtschaft des Landes Nordrhein -Westfalen“. Testing Laboratory and Dates Bayer CropScience AG - Development - Residues, Oper ator and Consumer Safety,

D-40789 Monheim, GER, conducted the work during the period of February to March 2006. Study completion date: 2006-03-22

Report MR-06/034 provides a method description and validation data f or the method 00986 that was developed for the determination of BYI08330 and its metabolite BYI08330-enol in soil. The objective of this work was to describe and to validate the residue an alytical method 00986 by means of recovery tests in two soils spiked with each of the compounds listed abov e. The method is the recommended enforcement method for monitoring of BYI08330 residues in soil.

Principle of the method: Residues of BYI08330 and the metabolite BYI08330-en ol are extracted from approx. 25 g of soil or sedim ent using an acidic extraction solution in the presence of cysteine hydrochloride and utilizing microwave extraction. The extraction solvent consists of a mi xture of water (containing 8 g/L L-cysteine hydroch loride), acetonitrile, ethyl acetate and formic acid. An ali quot of the final extract is analyzed by LC-MS/MS. Possible matrix effects are eliminated by using matrix stand ard solution. Identification and quantification of the test items is done by high performance liquid chromatogr aphy using MS/MS detection in the Multiple Reaction Monitoring (MRM) mode using instrumentation and con ditions presented in the following table.

HPLC System e.g. HP 1100 HPLC Column e.g. Luna 3μ C8(2) 100A, size 50 mm x 2 mm, Part No. 00B4248-B0, Phenomenex,

63741 Aschaffenburg, GER Injection Volume 20 μL

Solvent A: Milli-Q water/acetic acid (1000/5, v/v) Solvent B: Acetonitrile Solvent Isocratic Pump for flushing the interface: Milli-Q water/acetonitr ile/acetic acid (500/500/1, v/v/v) Oven Temperature: 40°C Flow rate (column): 0.2 mL/min Flow rate (into MS): 0.2 mL/min Gradient: Time [min] 0 2.9 3 5 7 8.1 11 11.1 13
% A 85 85 70 55 55 35 35 85 85 HPLC Method

% B 15 15 30 45 45 65 65 15 15 MS/MS System e.g. API 4000 with turbo-ionspray inte rface mass selective detector (MS/MS) (Perkin

Elmer Sciex Instruments, Weiterstadt, GER)– 34 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

BYI0833

0 m/z 302 BYI08330

m/z 216 BYI08330-enol

m/z 270 BYI08330-enol m/z 216

Q1 Mass [amu] Q3 Mass [amu] Dwell [msec] Ionisation Mode 374.1 301.8 250 TIS+ 374.1

215.9 250 TIS+ 302.2

269.7 250 TIS+ 302.2

215.8 250 TIS+ DP [V] CE [V] CXP [V] IS [V] EP [V] 51

25 10 5400 10 51

47 16 5400 10 71

29 8 5400 10 71

37 10 5400 10 MS/MS operating parameters

CAD CUR GAS 1 GAS 2 Turbo gas Temp. [°C] 6 10 70 70 650 6

10 70 70 650 6

10 70 70 650 6

10 70 70 650 Retention time BYI08330 (m/z 302): approx. 9.9 min

BYI08330 (m/z 216): approx. 9.9 min BYI08330-enol (m/z 270): approx. 7.2 min BYI08330-enol (m/z 216) : approx. 7.2 min

IS: Ion Spray Voltage EP: Entrance Potential CXP: C ollision Cell Exit Potential DP: Declustering Potential CE: Collision Energy TIS+: Turbo Ion Source in positive ion mode, i.e. p roduction of positive ions

Limit of quantification (LOQ) and Limit of Detectio n (LOD) : The limit of quantification of the method is 5 μg/k g for BYI08330 and BYI08330-enol. The LOD was set t o 1/3 of the LOQ (LOD = 1.5 μg/kg).

Calibration : Solvent standard solutions and matrix-mixed standar d solutions containing BYI08330 and BYI08330-enol were measured in a concentration range of 0.5 to 50 μg/L corresponding to a concentration in soil of 2 to 200 μg/kg. In this concentration range, the mass spectr ometric detector showed linear correlation between concentration and peak area for BYI08330 and BYI083 30-enol (a correlation coefficient of greater than 0.999 for all tests). The slope values of the linearity c urves show no matrix effects for BYI08330 m/z 302, BYI08330 m/z 216, BYI08330-enol m/z 270 and BYI08330-enol m/ z 216.

Specificity : The blank values in all control samples were below 1.5 μg/kg (<1/3 x LOQ), demonstrating that no background level of BYI08330 and BYI08330-enol was present in the two test systems.

Confirmatory method : Since HPLC-MS/MS and validation of two MRMs already offers maximum selectivity, i.e. specificity, a confirmatory procedure was considered to be unneces sary.

Validation : The method was validated with a silt soil (Höfchen) and a sandy loam soil (Laacher Hof) by analyzing c ontrol samples and control samples spiked with BYI08330 an d BYI08330-enol prior to the extraction procedure a t the limit of quantification of 5μg/kg as well as 10 times this limit (50 μg/kg). The soils were spiked with a mixture of BYI08330 and BYI08330-enol. For method v alidation, 20 recovery experiments were conducted. Each sample was injected into the HPLC instrument.– 35 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Spiking levels as well as recoveries and relative s tandard deviations (RSD) are presented in tables B. 5.3.1-1 to B.5.3.1-4. Spiking levels are expressed as conce ntration of test item in wet soil. The listed data demonstrate the successful validation of the analyt ical method 00986 for the determination of BYI08330 and its metabolite BYI08330-enol in soil.

TABLE B.5.3.1-1. Validation data for BYI08330 (m/z 302) in soils using the method 00986

Spiking [μg/kg] Soil Single Values [%] Mean [%] RSD

[%] 5 Höfchen 86 87 88 87 84 86 1.6
5 Laacher Hof 93 92 95 94 92 93 1.4 Mean of all 5 μg/kg single values 90 4.3 50 Höfchen 98 96 100 97 99 98 1.8
50 Laacher Hof 104 102 103 104 106 104 1.3 Mean of all 50 μg/kg single values 101 3.3 Mean of all Höfchen samples 92 6.8 Mean of all Laacher Hof samples 98 5.7

Overall mean 95 7.0

TABLE B.5.3.1-2. Validation data for BYI08330 (m/z 216) in soils using the method 00986

Spiking [μg/kg] Soil Single Values [%] Mean [%] RSD

[%] 5 Höfchen 86 88 87 87 87 87 0.9 5 Laacher Hof 91 90 95 92 92 92 2.1 Mean of all 5 μg/kg single values 89 3.6 50 Höfchen 96 94 97 96 94 95 1.5 50 Laacher Hof 104 102 100 103 105 103 1.9 Mean of all 50 μg/kg single values 99 4.2 Mean of all Höfchen samples 91 5.2 Mean of all Laacher Hof samples 97 6.1

Overall mean 94 6.5

TABLE B.5.3.1-3. Validation data for BYI08330-enol (m/z 270) in soils using the method 00986

Spiking [μg/kg] Soil Single Values [%] Mean [%] RSD

[%] 5 Höfchen 90 89 87 88 87 88 1.7 5 Laacher Hof 99 95 98 97 90 96 3.7 Mean of all 5 μg/kg single values 92 5.0 50 Höfchen 94 93 96 94 94 94 1.1 50 Laacher Hof 98 98 95 99 99 98 1.6 Mean of all 50 μg/kg single values 96 2.4 Mean of all Höfchen samples 91 3.6 Mean of all Laacher Hof samples 97 2.9

Overall mean 94 4.3

RSD: relative standard deviation

TABLE B.5.3.1-4. Validation data for BYI08330-enol (m/z 216) in soils using the method 00986

Spiking [μg/kg] Soil Single Values [%] Mean [%] RSD

[%] 5 Höfchen 92 91 89 90 88 90 1.8– 36 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

5 Laacher Hof 97 95 95 95 89 94 3.4 Mean of all 5 μg/kg single values 92 3.6 50 Höfchen 93 93 97 95 95 95 1.9 50 Laacher Hof 96 96 94 97 100 97 2.4 Mean of all 50 μg/kg single values 96 2.3 Mean of all Höfchen samples 92 3.4 Mean of all Laacher Hof samples 95 3.1

Overall mean 94 3.6

RSD: relative standard deviation

Conclusion : The method 00986 (report MR-06/034) was developed f or determination of BYI08330 and its metabolite BYI08330-enol in soil with an estimated limit of qu antification of 5 μ g/kg. This method was successfully validated according to the European requirements (9 6/46/EC of 16 July 1996 amending Directive 91/414/EEC), the Guidance Document on Residue Analy tical Methods (SANCO/825/00 rev.7 of March 17, 2004) and BBA Guideline for Residue Analytical Meth ods of July 21, 1998: - the overall mean recovery of the method, which was determined in two soils at le vels of 5 and 50 μg/kg each, was 95% for BYI08330 m /z 302 (RSD: 7.0%), 94% for BYI08330 m/z 216 (RSD: 6.5 %), 94% for BYI08330-enol m/z 270 (RSD: 4.3%) and 94% for BYI08330-enol m/z 216 (RSD: 3.6%). The repeatability expressed as the relative standard deviation (RSD) was lower than 7%. The method was found to be acceptable for monitoring of BYI08330 residues in soil.

Reference: In house laboratory validation of an analytical met hod for the determination of BYI08330 and its metabolites BYI08330-enol, BYI0 8330-ketohydroxy and BYI08330-MA-amide in soil and sediment by LC-MS/MS Author(s), year: Netzband, D. J.,2006 Report/Doc. number: RAFNX012 Guideline(s): US-EPA Test Guidelines OPPTS 850-7100 (1996), OPPTS 860.1340 (1996) GLP: Yes
Deviations: None Validity: Study considered acceptable

Material and methods: Test substances: Spirotetramat, spirotetramat-enol, spirotetramat-ketohydroxy, spirotetramat-MAamide (all unlabelled)

Internal standard: 13 C 3 -Spirotetramat, 13 C 3 -spirotetramat-enol, 13 C 3 -spirotetramat-ketohydroxy, 13 C 3 - spirotetramat-MA-amide
Test system: Two soil samples (North Rose, New York , study nr. MEFNY004, Lenz, 2006a, and Ephrata, Washington, study nr. MEFNY003, Lenz, 2006 d) and one sediment (Columbia, SC);
13 replicates for each soil, 7 replicates for the s ediment
Spiking: 5 and 25 μg kg -1 of each analyte separately

Internal laboratory validation: An Intern laboratory validation (ILV) on the method has been performed with the soils ‘Höfchen’ and ‘Laacherhof’, spiking level not mentioned in the report
Sample processing: Analytical method FN-002-S05-02 (updated to FN-002-S05-03): 20 g of soil are mixed with [25 mL of a solution of ethyl acetate (5 %, v/v) and cysteine hydrochloride (8 g L -1 ) in water, 0.5 mL formic acid, 25 mL acetonitrile] , spiked with 1 ml of 13 C internal standard solution and extracted by micro wave extraction– 37 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

(70 °C, 30 min). Extraction (without addition of in ternal standard) is repeated once. Following extraction samples are filtered (0.45 μm) to await LC-MS/MS analysis.
Analytical techniques: LC-MS/MS (screening for spir otetramat-enol dimer 1 and 2 possible)

Limit of detection (LOD): Defined as: (standard deviation (spiking 5 μg kg -1 ) × t 0.99 ) + average apparent residue in untreated control;

where t 0.99 is the one-tailed t-statistic at the 99 % confiden ce level of n-1 replicates (i.e. 2.68 in case of n = 13 and 3.14 in case of n = 7 )

Limit of quantification (LOQ): Defined as: (standard deviation (spiking 5 μg kg -1 ) × 5) + average apparent residue in untreated control

Findings: Calibration solutions of each analyte are stable ov er a minimum of 4 months when refrigerated.

TABLE B.5.3.1-5. Mean recover and relative standar d deviation (RSD) of analytes of freshly spiked soil and sediment samples. Soil Sediment Analyte Spiking level [μg kg -1 ] Overall mean

[%] RSD [%] Overall mean [%] RSD [%]

5 101 3.6 106 3.1 Spirotetramat 25 106 1.6 108 3.2

5 93 16.9 108 16.2 Spirotetramat-enol 25 84 11.7 84 3.9
5 98 6.9 110 5.6 Spirotetramat- ketohydroxy 25 103 5.7 105 7.2

5 105 3.3 117 3.4 Spirotetramat-MA-amide 25 103 3.5 108 5.9

TABLE B.5.3.1-6. Calculated LODs [μg kg -1 ] and LOQs [μg kg -1 ] for spirotetramat, spirotetramat-enol, spirotetramat-ketohydroxy and s pirotetramat-MA-amide in soil and sediment samples.
Matrix Analyte Calculated LOD [μg kg -1 ] Calculated LOQ [μg kg -1 ] Spirotetramat 0.5 0.9 Spirotetramat-enol 0.5 0.9 Spirotetramat-ketohydroxy 2.1 3.9 Soil

Spirotetramat-MA-amide 0.9 1.7

Spirotetramat 0.5 0.8 Spirotetramat-enol 0.6 1.0 Spirotetramat-ketohydroxy 2.7 4.4 Sediment

Spirotetramat-MA-amide 1.0 1.5

TABLE B.5.3.1-7. Results (mean recover and relativ e standard deviation, RSD) of the ILV using the soils ‘Höfchen’ and ‘Laacherhof’ freshly spiked with each analyte (spiking level not mentioned in the report).

Höfchen Laacherhof Analyte Overall mean [%] RSD [%] Overall mean [%] RSD [%]
Spirotetramat 94 1.8 92 1.4 Spirotetramat-enol 91 2.8 91 1.7– 38 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Höfchen Laacherhof Analyte Overall mean [%] RSD [%] Overall mean [%] RSD [%]
Spirotetramatketohydroxy 85 1.7 83 2.8

Spirotetramat-MA-amide 89 13.7 94 13.1

Conclusion: The analytical method FN-002-S05-02 (updated to FN- 002-S05-03 after revision) is suitable for the determination of spirotetramat and the metabolites spirotetramat-enol, spirotetramat-ketohydroxy and spirotetramat-MA-amide in soil and sediment by LC-M S/MS. The average recoveries for all analytes were
between 70 and 120 % with a relative standard devia tion (RSD) of less than 20 %. The method was shown capable of quantifying spirotetramat and its metabo lites at the targeted LOQ of 5 μg kg -1

.– 39 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.3.2 Water (Annex IIA 4.5, Annex IIIA 5.5)

Report: KIIA 4.5/01; Brumhard, B., 2006 (MR-131/03) Title: Analytical Method 00836 for the Determinatio n of BYI08330 and BYI08330-

enol in Drinking and Surface Water by HPLC-MS/MS an d HPLC-UV Report No & Edition No MR-131/03 (Method No. 00836)

M-250638-01-1 Guidelines: EC Guidance Document on Residue Analyti cal Methods, SANCO/825/00 rev.7 of

March 17, 2004. BBA Guideline: Residue Analytical M ethods for Post-Registration Control Purposes of July 21, 1998. Commission Direc tive 96/46/EC amending Council Directive 91/414/EEC of 16 July 1996. US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 GLP Fully GLP compliant - laboratory certified by G erman “Ministerium für Umwelt,

Raumordnung und Landwirtschaft des Landes Nordrhein -Westfalen“. Testing Laboratory and Dates Bayer CropScience AG - Development - Residues, Oper ator and Consumer Safety,

D-40789 Monheim, GER, conducted the work during the period of February 2004 to April 2005. Study completion date: 2005-4-29

Report MR-131/03 provides a method description and validation data for the method 00836 that was developed for the determination of BYI08330 and the metabolite BYI08330-enol in drinking and surface water. The objective of this work was to describe a nd to validate the residue analytical method 00836 by means of recovery tests in drinking and surface wat er spiked with each of the compounds listed above. The method is the recommended enforcement method for mo nitoring of BYI08330 residues in drinking and surface water.

Principle of the method: Water samples are analysed by direct injection into an HPLC instrument without separate extraction and clean-up steps. Identification and quantification o f the test items is done by high performance liquid chromatography using MS/MS detection in the Multipl e Reaction Monitoring (MRM) mode or UV detection, using instrumentation and conditions presented in t he following table.– 40 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

HPLC-MS/MS System e.g. HP 1100 with Quattro II with electrospray inte rface and mass selective detector (MS/MS) Waters GmbH, 65760 Eschborn, GER HPLC Column Synergi 4μ Fusion-RP 80, length 150 mm; i.d. 4.6 mm, article no. OOF-4424-EO; Phenomenex, 63768 Hosbach, GER Sample preparation HPLC-MS/MS Before analysis 20% of acetonitrile is added to the surface water samples. Additionally, acetic acid is added to a final conce ntration of 0.1 mL/L. These subsamples are directly injected into the HPLC inst rument (HPLC-UV). Before analysis the water samples were adjusted to pH 3 with o-phosphoric acid. Additionally, 20% acetonitrile was added to the sam ples. These solutions were directly injected in HPLC instrument. Injection Volume 220 μL (if required it can be adap ted to the concentration to be measured) Solvent A: Milli-Q water/acetonitrile/acetic acid ( 900/100/0.1, v/v/v) Solvent B: Acetonitrile/acetic acid (1000/0.1, v/v) Oven Temperature: 40°C Flow rate (column): 1 mL/min Gradient: Time [min] 0 1 7 10 10.1
% A 75 75 10 10 75 HPLC Method

% B 25 25 90 90 25 MS/MS operating parameters Bath gas: 400 L/h nitrogen 5.0 Nebulizer gas: 12 l/h nitrogen 5.0 Reactant gas: Argon 5.0 (3.5 bar)

Test item Detec tion

time Principle Dwell

[sec] Cone

[V] Collision energy [eV] Mass of parent ion [amu] Mass of product

ion [amu]

BYI08330 5.0-9.0 ESP+ 0.5 42 32 374.23 215.95 BYI08330- enol 5.0-9.5 ESP+ 0.5 45 28 302.30 216.00

Retention time BYI08330: approx. 8 min BYI08330-enol: approx. 6 min

HPLC-UV System e.g. HP 1090 with HP1100 Series Wave length Detector Agilent, 40880 Ratingen, GER HPLC Column Synergi 4μ Fusion-RP 80, length 150 mm; i.d. 4.6 mm, article no. OOF-4424-EO; Phenomenex, 63768 Hosbach, GER Injection Volume 250 μL (if required it can be adap ted to the concentration to be measured) HPLC Method Solvent A: Milli-Q water adjusted to pH 3 with o-phosphoric acid Solvent B: Acetonitrile Oven Temperature: 40°C Flow rate (column): 1 mL/min Gradient: Time [min] 0 1 7 10 10.1
% A 75 75 10 10 75 % B 25 25 90 90 25 UV operating parameter Wavelength: 210 nm

Retention time BYI08330: approx. 8.5 min BYI08330-enol: approx. 7 min

Limit of quantification (LOQ):– 41 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

The limit of quantification for BYI08330 and BYI083 30-enol in surface water and drinking water is 0.05 μg/L, and the limit of detection is 0.02 μg/L, respective ly.

Calibration: In surface water the mass spectrometric detector sh owed linear response in the concentration range of approx. 0.04 μg/L to 12 μg/L for BYI08330 and BYI08 330-enol with a correlation coefficient of > 0.9998 . The UV detector showed linear response in surface water for both analytes in the concentration range of ap prox. 5 μg/L to 1000 μg/L with a correlation coefficient of 1.0000. The MS/MS detection of BYI08330 and BYI08330-enol is affected by the matrix. The peak a rea of BYI08330 and BYI08330-enol in a surface wate r sample containing 0.5 μg/L was reduced to approx. 7 3% and 69%, respectively, of the corresponding peak area in Milli-Q-water. The UV detection of BYI08330 and BYI08330-enol is not affected by the matrix.

Specificity: The blank values of all control samples were below 0.3 x LOQ.

Repeatability: Repeatability testing of MS/MS detection of BYI0833 0 and BYI08330-enol in drinking and surface water samples spiked at concentrations of 0.05 μg/L and 0 .5 μg/L yielded a relative standard deviation for t he peak area in the range of 1.4% and 6.5%, respectively. T he relative standard deviation for the retention ti me was < 0.2% for both analytes at both spiking levels. Repeatability testing of UV detection of BYI08330 a nd BYI08330-enol in surface water samples spiked at concentrations of approx. 5 μg/L and 1000 μg/L yiel ded a relative standard deviation for the peak area in the range of 0.4% and 3.1%. The relative standard devia tion for the retention time was < 0.1 % in each cas e.

Confirmatory method: Since HPLC-MS/MS already offers maximum selectivity , i.e. specificity, a confirmatory procedure was considered to be unnecessary.

Validation using MS/MS-detection: For method validation surface water from the river Rhine sampled in Leverkusen-Hitdorf and drinking wa ter sampled in Monheim am Rhein, Bayer CropScience AG, building 6610, was used. For method validation surface and drinking water samples were spiked with BYI08330 and BYI08330-enol at 0.05 μg/L and at 0.5 μg/L. These test solutions were injected ten times each into the HPLC-MS/MS instrument. The peak areas for BYI08330 and BYI08330-enol were determined and are listed in table B.5.3.2-1 and B.5.3.2-2. The relative standard deviation for the peak area of BY I08330 in surface water was 3.4% (0.05 μg/L) and 1. 4% (0.5 μg/L), and in drinking water 6.5% and 5.4%, re spectively. The relative standard deviation for the retention time was always < 0.1%. The relative stan dard deviation for the peak area of BYI08330-enol i n surface water was 2.7% (0.05 μg/L) and 1.7% (0.5 μg /L), and in drinking water 6.1% and 2.6%, respectiv ely. The relative standard deviation for the retention t ime was < 0.2%, always

TABLE B.5.3.2-1. Validation data for BYI08330 and B YI08330-enol in surface water using the method 00836 (MS/MS-detection)
Peak area Retention time Spiking level (μg/L) Individual values (area counts) Mean

(area counts) RSD

(%) Mean

(min) RSD (%)

BYI08330
0.05 88 / 89 / 84 / 88 / 81

81 / 84 / 84 / 83 / 83 85 3.4 7.6 <0.1

BYI08330
0.5 809/824/811 / 794/821

806 / 797 / 799 / 790 / 798 805 1.4 7.6 <0.1

BYI08330-enol 83 / 80 / 81 / 79 / 82 81 2.7 5.9 0.2– 42 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Peak area Retention time Spiking level (μg/L) Individual values (area counts) Mean

(area counts) RSD

(%) Mean

(min) RSD (%)

0.05 85 / 84 / 82 / 79 / 79
BYI08330-enol
0.5 847/821 / 846/859/839

842/855/831 / 81 5/851 841 1.7 5.9 0.1

TABLE B.5.3.2-2. Validation data for BYI08330 and B YI08330-enol in drinking water using the method 00836 (MS/MS-detection)
Peak area Retention time Spiking level (μg/L) Individual values (area counts) Mean

(area counts) RSD

(%) Mean

(min) RSD (%)

BYI08330
0.05 38 / 35 / 38 / 38 / 41

36 / 34 / 37 / 33 / 35 37 6.5 8.3 0.1

BYI08330
0.5 333/329/323/322/312

31 1 / 302 / 297 / 293 /

282 310 5.4 8.3 <0.1

BYI08330-enol
0.05 38 / 35 / 39 / 35 / 32

38 / 37 / 36 / 35 / 39 36 6.1 6.7 0.1

BYI08330-enol
0.5 340 / 329 / 343 / 337 / 326

324/323/322/323/319 329 2.6 6.7 0.1

Because of the direct measurement of spiked samples without separate extraction and clean-up steps, it is not possible to determine recovery rates and an est imate of the accuracy of the analytical technique w as made by an assessment of the linearity of matrix ca libration and by determination of the reproducibili ty of sample analysis. However, for additional demonstrat ion of the reliability of the method, the validatio n samples were evaluated like recovery rates. From th e single peak area of each sample listed, the percentages in Table 4.5-1 to Table 4.5-4 were calc ulated relative to the corresponding mean area, whi ch
was set at 100%.

TABLE B.5.3.2-3. Percentages of BYI08330 and BYI083 30-enol found in the spiked surface water samples using method 00836 (MS/MS-detection) Percentage found

Spiking level (μg/L) Individual values (%) Mean [%) RSD

(%)
BYI08330
0.05 104 / 105 / 99 / 104 / 96

96 / 99 / 99 / 98 / 98 100 3.4

BYI08330
0.5 101 / 102 / 101 / 99 / 102

100 / 99 / 99 / 98 / 99 100 1.4

BYI08330-enol
0.05 102 / 98 / 100 / 97 / 101

104 / 103 / 101 / 97 / 97 100 2.7

BYI08330-enol
0.5 101 / 98 / 101 / 102 / 100

100 / 102 / 99 / 97 / 101 100 1.7

The MS/MS detection of BYI08330 and BYI08330-enol i s affected by the matrix. The peak area of BYI08330 in a surface water sample containing 0.5 μg/L was r educed to approx. 73% of the corresponding peak are a in Milli-Q-water and the peak area of BYI08330-enol in a surface water sample (0.5 μg/L) was reduced t o approx. 69% of corresponding peak area in Milli-Q-w ater. The matrix effect was determined by four inje ctions of each solution.– 43 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE B.5.3.2-4. Percentages of BYI08330 and BYI083 30-enol found in the spiked drinking water samples using method 00836 (MS/MS-detection) Percentage found

Spiking level (μg/L) Individual values (%) Mean [%) RSD

(%)
BYI08330
0.05 104 / 96 / 104 / 104 / 112

99 / 93 / 101 / 90 / 96 100 6.5

BYI08330
0.5 107 / 106 / 104 / 104 / 101

100 / 97 / 96/ 94 / 91 100 5.4

BYI08330-enol
0.05 104 / 96 / 107 / 96 / 88

104 / 102 / 99 / 96 / 107 100 6.1

BYI08330-enol
0.5 103 / 100 / 104 / 103 / 99

99 / 98 / 98 / 98 / 97 100 2.6

Validation using UV-detection: For method validation surface water samples of rive r Rhine were spiked with BYI08330 and BYI08330-enol at about 5 and 1000 μg/L. These test solutions were injected ten times each into the HPLC-UV instrumen t. The peak areas and retention times for BYI08330 and BYI08330-enol were determined and are listed in Table 16 and Table 17 of report MR-131/03. The rela tive standard deviation (RSD) for the peak area of BYI08330 in surface water was 1.6% (5 μg/L) and 0.4 % (1000 μg/L), respectively, and the RSD for the retention time was < 0.1 % for both concentrations. The relative standard deviation (RSD) for the peak area of BYI08330-enol in surface water was 3.1% (5 μg/L) and 0.6% (1000 μg/L), respectively, and the RSD fo r the retention time was < 0.1 % for both concentrati ons. Because of the direct measurement of spiked samples without separate extraction and clean-up st eps it is not possible to determine recovery rates and an estimate of the accuracy of the analytical techniqu e was made by an assessment of the linearity of mat rix calibration and by determination of the reproducibi lity of sample analysis. However, for additional demonstration of the reliability of the method, the validation samples were evaluated like recovery ra tes. From the single peak area of each sample the percen tages listed in table B.5.3.2-5 were calculated rel ative to the corresponding mean area, which was set as 10 0%.

TABLE B.5.3.2-5. Percentages of BYI08330 and BYI083 30-enol found in the spiked surface water samples using method 00836 (UV-detection) Percentage found Spiking level (μg/L) Individual values (%) Mean [%) RSD

(%)
BYI08330
5 99 / 99 / 99 / 98 / 102

101 / 102 / 101 / 99 / 99 100 1.6

BYI08330
1000 101 / 100 / 100 / 100 / 99

99 / 100 / 100 / 100 100 0.4

BYI08330-enol
5 97 / 100 / 98 / 100 / 100

107 / 101 / 103 / 96 / 98 100 3.1

BYI08330-enol
1000 100 / 101 / 100 / 100 / 99

99 / 100 / 100 / 100 / 100 100 0.6

Storage stability: For examination of the storage stability surface wa ter samples spiked separately with about 0.5 μg BYI08330/L and BYI08330-enol/L each were stored in a freezer at < -18 °C and in a refrigerator at < 6 °C for a period of about 4 months. Taking the pH-dependent hydrolytic stability of the two analytes into acco unt the water samples spiked with BYI08330 were adjusted to pH 3 and the water samples containing BYI08330- enol were kept at its natural pH (ca. pH 8). Prior to storage, all solutions were diluted with 20% of acetonitrile– 44 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

as outlined in earlier. A set of three samples was analyzed after 140 days of storage. The results sho wed that under freezer and refrigerator conditions BYI0 8330 and BYI08330-enol were stable for a storage pe riod of at least 140 days. The mean recoveries were in t he range of 92 to 107% for BYI08330 and in the rang e of 76 to 113% for BYI08330-enol.

Conclusion: The method 00836 (report MR-131/03) was developed f or determination of BYI08330 and its metabolite BYI08330-enol in surface and drinking water with an estimated limit of quantification of 0.05 μg/L. Th is method was successfully validated according to the European requirements (96/46/EC of 16 July 1996 amending Directive 91/414/EEC), the Guidance Docume nt on Residue Analytical Methods (SANCO/825/00 rev.7 of March 17, 2004) and BBA Guideline for Resi due Analytical Methods of July 21, 1998:

The method was assessed to be acceptable for monito ring of BYI08330 and BYI08330-enol residues in surface and drinking water.

However, some points need clarification: Method preparation (surface water): The notifier is asked to clarify: Were the samples spiked with active ingredient before or after filtr ation of the dry residue LC-MS/MS-detection : The calibration/linearity data for drinking water s hould be submitted. The described matrix effect for reducing the values to 73% and 69% respectively must be clarified in d etail. The notifier is requested to submit raw data. UV-detection : The calibration range should extend over a range to the lowest and highest nominal concentration at le ast ± 20%. For surface water the lowest measured concentr ation for calibration is 5.292 μg/L for BYI 8330 an d 5.133 μg/L for BYI 8330-enol respectively. Therefor e the LOQ of 5.0 μg/L is not covered by the calibra tion range according to the EU guideline SANCO 825/00. T his is of concern since all ecotoxicological studie s have been investigated with the LC-UV method.

B.5.3.3 Sediment (Annex IIA 4.6, Annex IIIA 5.6)

Refer to document MR-06/034 from point 4.4 (KIIA 4. 4/01).

B.5.3.4 Air (Annex IIA 4.7, Annex IIIA 5.7)

Not required or reviewed by the Canada PMRA.

Required according to Directive 91/414/EEC and eval uated by Austria (AGES) for EU.

Report: KIIA 4.7/01; Stupp, H.-P.; 2006 (MEF-06/119 ) Title: Method for the Determination of BYI08330 in Air by HPLC-UV and

Confirmation by DAD (UV Spectra Match) Report No & Edition No MEF-06/119 (Method No. 01015)

M-274784-01-1 Guidelines: EU Directive 91/414/EEC Annex II (Part A, Section 4.2), as amended by

Commission Directive 96/46/EC. EC Guidance document on residue analytical methods, SANCO/825/00 rev. 7 17/03/04. US-EPA Residue Chemistry Test Guideline OPPTS 860.1 340 GLP Fully GLP compliant - laboratory certified by G erman “Ministerium für Umwelt,

Raumordnung und Landwirtschaft des Landes Nordrhein -Westfalen“. Testing Bayer CropScience AG, Metabolism and Enviro nmental Fate, D-40789 Monheim,– 45 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Laboratory and Dates GER,

conducted the study during the period of February t o March 2006. Study completion date: 2006-06-06 Validity Acceptable for the determination of spirot etramat (BYI08330)

The objective of this study was to describe and to validate the residue analytical method by means of recovery tests in ambient and with warm, humid air (approx. 35°C, 80% relative humidity) spiked with BYI08330. In addition, a test with the major metabo lite BYI08330-enol was included and reported.

Test material: Air at elevated (35 °C, 80% relative humidity) clim atic conditions.

Spiked analyte: Spirotetramat

Analyte(s) determined as: Spirotetramat

Principle of the method: The air to be analysed is passed through commercial sealed cartridges at a flow rate of about 2 L/min for 6 hours (total volume of 720 L). The sampling tubes c omprise two portions of porous adsorbent polymer (Tenax®) separated with glass wool: the front porti on (portion A, 100 mg adsorption material) is inten ded for collection of the analyte and the back portion (por tion B, 50 mg adsorption material) allows control o f breakthrough. Following sampling, the tubes are dir ectly processed or are closed with plastic caps and are kept cool. The glass wool separating both layers an d the upper glass wool facing the inlet of the tube are processed together with the front portion. The indi vidual layers are removed from the glass tube and p assed into separate 10-mL vials with snap cover. The glas s tube is rinsed with 4 mL of acetonitrile. The fro nt portion is mixed with these 4 mL of solvent and the back portion is mixed with 4 mL of acetonitrile, a lso. The test item is extracted from the adsorption material by shaking a laboratory shaker (about 300 rpm) for 30 minutes. For HPLC analysis (i.e. using UV detection with external standard) of the upper LOQ samples t he extracts were diluted 1:100 (v/v) with acetonitrile prior to injection in order to be in the same conc entration range for analysis quantified by liquid chromatogra phy. Instrumentation and conditions are presented i n the following table.– 46 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

HPLC System e.g. Agilent 1100 with DA-detector and stationary mixer (vol. ca 1 mL) Detection wavelength at 254 nm (BW 8 nm), reference 550 nm (BW 80 nm). HPLC Column e.g. Purosphere Star 18e Fa. Merck 5314 55; length = 250 mm, inner diameter = 4.6 mm, particle size =5 μ m. Pre-column: Purosphere RP 18 (Lichrocart 4-4, Fa

. Merck) Injection Volume 100 μ L (samples were injected in duplicate) HPLC Method Solvent A: 0.01% acetic acid in water Solvent B: Acetonitrile Oven Temperature: 25°C Flow rate (column): 1.0 mL/min Linear gradient: Time (min) %A %B
0.0 100 0
5.0 100 0
45.0 40 60
55.0 0 100
60.0 0 100
65.0 100 0 70.0 100 0 (post time 10 min) Retention time of BYI08330: 49.1 min

Findings: Specificity: Interferences in blank samples of max. 0.41 μg/m 3 [i.e. 4.1% of 10 μg/m 3 (LOQ)] were found which is acceptable. No confirmatory technique is n ecessary since spirotetramat is successfully valida ted in soil and water matrices.

Calibration/Linearity: The detector response was linear from 0.54 μg/mL t o 5.4 μg/mL (5 levels, r 2 = 0.9987). Samples of high spiking concentration were diluted 1:100 prior to injection.

LOQ: 10 μ g/m3 air.

Accuracy: Mean recoveries are between 70-110 % at each spiki ng level with a RSD < 20 %.

Breakthrough behaviour: No breakthrough was observed at both spikings (10 and 1000 μg/m 3 ).

Validation data are summarised in table B.5.3.4-1.

Spiked analyte: Spirotetramat-enol

Stability of spirotetramat-enol (BYI08330-enol) ads orbed onto 5 different materials (Tenax, XAD-2, Cha rcoal, Florisil and Silica Gel) was tested under same cond itions as described for spirotetramat (BYI08330). Desorption recoveries at a spiking of 10 μg/m 3 are either too low (20 and 33.1% mean recoveries) or non detectable due to complete degradation.– 47 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

TABLE 5.3.4-1: Validation data for spirotetramat (B YI08330) residues in air
References Determined

analyte Detection

method Matrix LOQ [μg/m

3 ] Spiking

level [μg/m 3 ] Mean

recovery [%] RSD

[%] n

elevated temperature and humidity (35°C and 80% RH)

10 105.7 3.2 5 Stupp, H.- P.; 2006(MEF06/119) BYI08330 HPLC-UV

Air 10

1000 105.4 1.22 5

Conclusion: The method is successfully validated for the determ ination of spirotetramat (BYI08330) in air. Accordi ng to Section Toxicology an AOEL systemic of 0.05 mg/kg b w/d was set which results in a value of 0.015 mg/m 3 air (equivalent to 15 μg/m 3 ) for the concentration C with respect to the calcu lation of guidance document 825/00. This concentration is covered by the LOQ se t at 10 μg/m 3

.

Adsorption tubes containing about 20 μg/m 3 BYI08330 (i.e. 2 times LOQ) were stable in a refri gerator at about +2/+6 °C and in a deep-freezer at about -21/- 24 °C for 8 days after sampling.

The method is not suitable for determination of the main degradation product BYI08330-enol which is no t included in the current residue definition in air.

B.5.4 Analytical methods for the determination or r esidues in body fluids and tissues

(Annex IIA 4,8; Annex IIIA 5.8)

Not applicable, as compounds are not classified as toxic (T, T+).– 48 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.5 Evaluation and assessment

The analytical methods provided for the analysis of the technical material and environmental samples h ave been assessed to be fully validated and acceptable.

B.5.5.1 Analytical methods for technical active sub stance and formulation analysis

Analytical methodology is available for the determi nation of the active substance and the impurities i n the technical material.

All methods are fully validated. No CIPAC method ex ists at the time of evaluation.

Table 5.5.1-1: Summary of analytical methods for t echnical active substance and formulation analysis Matrix Analyte Type of method Validation References
Technical active substance Spirotetramat HPLC-UV
Method AM001104MP1 Full Hake, 2004

HPLC-UV AM001204MP2 Full Hake, 2006

GLC (Headspace)
Method AM003406MP1 Full Kraemer, 2006 Technical active substance Impurities

Karl Fischer titration Internationally

accepted method CIPAC MT 30.5

Technical active substance Formulation HPLC-UV Method AM002104MF1 Full Odenahl 2005

B.5.5.2 Analytical methods for residues

The submitted methods enable the enforcement of the following relevant residue definitions and limits (at the time of evaluation).

Table 5.5.2-1: Overview of accepted residue analyt ical methods Matrix type/ crop group Primary Method Analyte Confirmatory method Independent Lab

Validation (if appropriate) Hope (cone dried) Avocado (fruit) Orange (fruit) Potato (tuber) Tomato (fruit) Schöning R., Willmes J. (2008) LC-MS/MS BYI08330

BYI08330-enol BYI08330- ketohydroxy BYI08330- monohydroxyBYI08330-enolglucoside Not necessary Not yet available

Animal origin Freitag, Th., BYI08330-enol Not neces sary Bacher R; 2006– 49 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Matrix type/ crop group Primary Method Analyte Confirmatory method Independent Lab

Validation (if appropriate) Wolters, A.; 2006 LC-MS/MS Soil Brumhard B.; 2006 LC-MS/MS Spirotetramat Not necessary –

Drinking water Spirotetramat Not necessary –
Surface water Brumhard B.; 2006 LC-MS/MS Spirotetramat Not necessary – Air Stupp H.-P.; 2006 LC-UV Spirotetramat Stupp H.-P.;
2006 LC-DAD (not necessary) –

Table 5.5.2-2: Summary of analytical methods cover ing relevant residue definitions and limits Matrix Residue definition LOQ Residue limit

Avocado (fruit) Orange (fruit) Potato (tuber) Tomato (fruit) 0.01 mg/kg for each analyte

Hope (cone dried) BYI08330 and the 4

metabolites BYI08330-enol, BYI08330-ketohydroxy, BYI08330-monohydroxy, and BYI08330 enol-glucoside, all metabolites expressed as BYI08330.

0.1 mg/kg for each analyte Proposed MRLs in mg/kg:

Tomatoes 2.0 Peppers 2.0 Aubergines (egg plants) 2.0 Cucumbers 0.2 Courgettes 0.2
Melons 0.2 Pumpkins 0.2 Watermelons 0.2 Flowering brassica 1.0 Brussels sprouts 0.3 Head cabbage 0.5 Kale 2.0 Kohlrabi 2.0 Lettuce 5.0 Hops 10.0

Milk 0.005 mg/kg BYI08330-enol calculated and expressed as BYI08330 parent equivalents Proposed MRLs in mg/kg:

Milk

Meat and egg BYI08330 and BYI08330-enol

0.01 mg/kg BYI08330-enol calculated and expressed as BYI08330 parent Proposed MRLs in mg/kg:

Liver, meat Meat by products (except liver)– 50 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Matrix Residue definition LOQ Residue limit equivalents

Soil Spirotetramat 5 μg/kg LC50 earthworm >1 g/kg s oil dry weight NOEC is not required by section ecotoxicology.

Drinking water Spirotetramat 0.05 μg/L 0.1 μg/L EU drinking water limit Surface water Spirotetramat 0.05 μg/L NOEC Chironom us riparius < 0.53 mg/L LC50 Chironomus riparius 1.30 mg/L Air Spirotetramat 10 μg/m3 15 μg/m3 based on a prop osed AOELsystemic of 0.05 mg/kg bw/day Body fluids Not necessary, the active substance is not classified as toxic or highly toxic– 51 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

B.5.6 References relied on

Annex point/ reference number Author(s) Year Title Source (where different from

company) Company, Report No GLP or GEP status (where relevant)

Published or not Data Protection Claimed

Y/N-R/NR Owner

Annex II Data and Information
KIIA 4.2.1 /01 Hake, G. 2004 Spirotetramat (BYI08330) - Assay of Technical Grade Active Ingredient - HPLC - External Standard Bayer CropScience AG, Report No.: AM001104MP1, Edition Number: M-084385-01-1 Date: 07.07.2004 Non GLP, unpublished Yes BCS

KIIA 4.2.1 /02 Hake, G. 2004 Validation of HPLC-method AM001104MP1 - BYI08330 Assay of technical grade active ingredient Bayer CropScience AG, Report No.: VB2-AM001104MP1, Edition Number: M-084403-01-1 Date: 07.07.2004, Amended: 09.11.2006 Non GLP, unpublished Yes BCS

KIIA 4.3 /18 Schöning, R.,

Willems, J., 2008 Analytical method 01084 for the determination of residues of spirotetramat (BYI 08330), BYI08330- enol, BYI08330-ketohydroxy, BYI08330-mono-hydroxy and BYI08330-enol-glucoside metabolites in/on plant material by HPLC-MS/MS Bayer CropScience AG, Report No.: MR-08/007, Edition Number: M-298287-01-1 Method Report No.: MR-08/007 Date: 17.08.2005, Amended: 02.06.2006 GLP, unpublished Yes BCS– 52 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Annex point/ reference number Author(s) Year Title Source (where different from

company) Company, Report No GLP or GEP status (where relevant)

Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIA 4.3 /08 KLimmek, S. 2005 BYI 08330-enol: Exami nation of the applicability of DFG merhod S 19 (extended and revised revision) for the determination of residues of BYI 08330-enol Specht & Partner Chemische Laboratorien GmbH, Hamburg, Germany Bayer CropScience AG, Report No.: BAY-0519, Edition Number: M-257512-01-1 Method Report No.: BAY-0519 Method Report No.: G05-0104 Date: 08.09.2005 GLP, unpublished Yes BCS

KIIA 4.3 /15 Freitag, T.;

Wolters, A. 2006 Analytical method 00969 for the determination of residues of BYI08330- enol in/on matrices of animal origin by HPLC-MS/MS Bayer CropScience AG, Report No.: 00969, Edition Number: M-265407-01-2 Method Report No.: MR-160/05 Date: 18.01.2006 GLP, unpublished Yes BCS

KIIA 4.3 /16 Bacher, R. 2006 Independent laboratory validation of BCS analytical method 00969 for the determination of residues of BYI08330- enol in materials of animal origin PTRL Europe, Ulm, Germany Bayer CropScience AG, Report No.: P613060584, Edition Number: M-269571-01-2 Method Report No.: P/B 964 G Date: 20.04.2006 GLP, unpublished Yes BCS

KIIA 4.4 /01 Brumhard, B. 2006 Analytical Method 00 986 for the determination of residues of BYI08330and BYI08330-enol in soil by HPLCMS/MS Bayer CropScience AG, Report No.: 00986, Edition Number: M-268586-01-1 Date: 22.03.2006 GLP, unpublished Yes BCS– 53 –

Spirotetramat – Volume 3, Annex B.5, Methods of Ana lysis April 2008

Annex point/ reference number Author(s) Year Title Source (where different from

company) Company, Report No GLP or GEP status (where relevant)

Published or not Data Protection Claimed

Y/N-R/NR Owner

KIIA 4.5 /01 Brumhard, B. 2006 Analytical method 00 836 for the determination of BYI08330 and BY08330-enol in drinking and surface water by HPLC-MS/MS and HPLC-UV Bayer CropScience AG, Report No.: MR-131/03, Edition Number: M-250638-01-2 Date: 01.08.2006 GLP, unpublished Yes BCS

KIIA 4.7 /01 Stupp, H. P. 2006 Method for the deter mination of BYI 08330 in air by HPLC-UV and confirmation by DAD (UV spectra match) Bayer CropScience AG, Report No.: 01015, Edition Number: M-274784-02-1 Method Report No.: MEF-06/119 Date: 06.06.2006, Amended: 07.11.2006 GLP, unpublished Yes BCS

Annex III Data and Information
KIIIA1 5.2.1 /01 Zitzmann, W. 2004 Determination of Spirotetramat in formulations - assay - HPLC, external standard Bayer CropScience AG, Report No.: AM002104MF2, Edition Number: M-068817-02-2 Date: 11.05.2004, Amended: 26.03.2007 Non GLP, unpublished Yes BCS

KIIIA1 5.2.1 /02 Odendahl, A. 2005 Validation of HPLC-method AM002104MF1 - determination of BYI 08330 in formulations - Bayer CropScience AG, Report No.: VB2-AM002104MF1, Edition Number: M-260838-02-2 Date: 14.11.2005 Non GLP, unpublished Yes BCS