ISOXAFLUTOLE (268) · Isoxaflutole was scheduled for evaluation as a new compound by 2013 JMPR at...

Isoxaflutole

1171

ISOXAFLUTOLE (268)

First draft prepared by Ms T. van der Velde-Koerts and K.M. Mahieu, Centre for Food, Prevention and Health Services, National Institute of Public Health and the Environment, The Netherlands

EXPLANATION

Isoxaflutole was scheduled for evaluation as a new compound by 2013 JMPR at the 44th session of the CCPR (2012).

Isoxaflutole is a synthetic compound of the isoxazole group of chemicals used as an herbicide. The mode of action of isoxaflutole is the inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD), thereby inhibiting pigment formation, and causing bleaching of the developing tissues of the target plants. Isoxaflutole controls a wide spectrum of grasses and broadleaf weeds by bleaching emerging or emerged weeds following herbicide uptake via the root system.

The Meeting received information from the manufacturer on identity, metabolism, storage stability, residue analysis, use pattern, residues resulting from supervised trials on sweet corn, chickpeas, glyphosate/HPPD tolerant soya beans, maize, sugar cane and poppy seed, fate of residue during processing, and livestock feeding studies.

IDENTITY

ISO common name: isoxaflutole

Chemical name

IUPAC: 5-cyclopropyl-4-(2-methylsulfonyl-4-trifluoromethylbenzoyl)-isoxazole

CAS: (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]-methanone

CAS Registry No: 141112-29-0

CIPAC No: 575

Synonyms and trade names:

RPA 201772; AE B197278; AE B197278; RPA 591428; 94 BCS-AH21981 IFT

Structural formula:

Molecular formula: C15H12F3NO4S

Relative Molecular weight:

359.3

Structure confirmed by:

UV-VIS (aq MeOH), IR (KBr pellet), 1H-NMR (250 MHz, CDCl3), 13C-NMR (62.9 MHz, CDCl3), EI-MS [Guesnet et al., 1994, M-189749-01-1]

PHYSICAL AND CHEMICAL PROPERTIES

Pure active ingredient, purity at least 99.7% w/w

Parameter Result References Guidelines/ method

Appearance: White granular powder [Cousin, 1994b, visual

CF3

SO2CH3O

NO

Isoxaflutole

1172


No characteristic odour Batch JYG 708, 99.7% w/w

M-213139-02-1]

Vapour pressure: 3.22 × 10–4 mPa at 20 °C (measured) 1.00 × 10–3 mPa at 25 °C (calculated) 7.71 × 10–3 mPa at 35 °C (measured) 7.90 × 10–2 mPa at 50 °C (measured) Technical material, batch 21ADM93, 98.7% w/w

[Cousin, 1994a, M-162435-01-1]

OECD 104 EC A4 gas saturation

Melting point: 140 ± 1 °C Batch JYG 708, 99.7% w/w

[Cousin, 1994b, M-213139-02-1]

EC A1 Differential calorimetric analysis

Octanol/water partition coefficient:

log Kow = 2.34 at 20 °C pH not indicated Batch JYG 708, 99.7% w/w

[Cousin, 1995b, M-162438-03-1]

OECD 117 EC A8 HPLC method

Solubility: Solubility depends on temperature. at 10 °C 0.0053 g ai/L in water (pH 6.8) at 20 °C 0.0062 g ai/L in water (pH 5.5) 0.0068 g ai/L in pH 5 buffer degradation in pH 9 buffer at 30 °C 0.0118 g ai/L in water (pH 5.9) Technical material, batch 21ADM93, 98.7% w/w Note: the solubility of a substance can be considerably affected by the presence of impurities. Solubility has not been tested for the pure active substance.

[Cousin, 1993c, M-162137-01-1]

OECD 105, EC A6 column elution method

at 20 °C acetone 293 g ai/L acetonitrile 233 g ai/L dichloromethane 346 g ai/L octanol-1 0.76 g ai/L ethyl acetate 142 g ai/L hexane 0.1 g ai/L methanol 13.8 g ai/L toluene 31.2 g ai/L

Technical material, batch 21ADM93, 98.7% w/w Note: the solubility of a substance can be considerably affected by the presence of impurities. Solubility has not been tested for

[Cousin, 1993c, M-162137-01-1]

OECD 105, EC A6 flask method

Isoxaflutole

1173


the pure active substance.

Specific gravity Density:

specific gravity 1.593 (20 °C/20 °C) density 1.590 g/cm3 at 20 °C Batch JYG 708, 99.7% w/w

[Cousin, 1994b, M-213139-02-1]

OECD 109 EC A3 pyknometer

Hydrolysis in water: at 25 ± 1 °C, in the dark, under sterile conditions, at an initial concentration of 3 mg ai/L Duplicate samples were removed at:

pH 5: 0, 3, 5, 7,10,12 and 14 days pH 7: 0, 3, 5, 7,16,19 and 24 hours pH 9: 0,1,1.5,2,2.5,3,4 and 24 hours.

The radioactivity balance ranged from 98.2 to 101.3% of the initial radioactivity. No volatile compounds were formed at any pH studied. Isoxaflutole was hydrolysed following a pseudo-first order kinetics, with half-life DT50 11.1 days at pH 5 20.1 hours at pH 7 3.2 hours at pH 9 [U-14C-phenyl]-Isoxaflutole (batch JKS 473, radiochem purity > 98% w/w) The only hydrolysis-product is IFT-DKN pH 5: 1.2–55% TAR at 0–14 days pH 7: 1.7–54% TAR at 0–24 hours pH 9: 2.2–95% TAR at 0–24 hours

[Corgier et al., 1994, M-162558-01-1]

EPA 161-1

Photolysis in water: at 25 ± 1 °C, under sterile conditions, at an initial concentration of 3.0 mg ai/L in pH 5 buffer with 1% v/v ACN, as a co-solvent. Duplicate samples were removed after 0, 16, 30, 40 and 54 hours of irradiation using a Xenon lamp at wavelengths > 290 nm at 612 W/m2. The radioactivity balance ranged from 97.3 to 99.9% of the initial radioactivity. A maximum of 0.11% of the initial radioactivity was recovered as volatile compounds at 54 hours. Isoxaflutole was photodegraded following pseudo-first order kinetics with half-life DT50 40 hrs [U-14C-phenyl]-isoxaflutole (batch JKS 473, radiochemical purity > 98% w/w)

[Corgier and Plewa, 1995, M-162794-01-1]

EPA 161-2

Isoxaflutole

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Several degradates were observed: IFT-BA, 0.9–2.8%TAR at 0–54 hrs, IFT-DKN, 0.0–2.7% TAR at 0–54 hrs RPA 205834, 0.0–1.7% TAR at 0–54 hrs Met 20 (MW 359), 0.0–16.8% TAR at 0–54 hrs Met 14 (MW 377), 0.0–9.3% TAR at 0–54 hrs

Dissociation constant: pH 4.6 at 25 ºC, 1% w/v aqueous suspension in water containing 2% (v/v) ACN No dissociation constant could be determined. The solubility of isoxaflutole in water containing 3% (v/v) ACN or MeOH was too low to use the potentiometric method. The UV spectra of isoxaflutole did not differ significantly with the pH and did not allow the determination of pKa using the spectrophotometric method. Technical materials Batch 21ADM93, 98.7% w/w Batch FP1308, 98.3% w/w

[Cousin, 1993b, M-162129-01-2]

OECD 112 EPA 63-10 EPA 63-12

Technical material, purity at least 97.5% w/w


Appearance: Yellow granular powder slight characteristic odour, acetic acid-like Batch 21ADM93, 98.7% w/w

[Cousin,1993a, M-162115-01-1]

EPA 63-2 EPA 63-3 EPA 63-4 visual

Yellow granular powder slight characteristic odour, acetic acid-like Batch FP1308 98.3% w/w

[Cousin,1993a, M-162115-01-1]

EPA 63-2 EPA 63-3 EPA 63-4 visual

Specific gravity Density:

Specific gravity 1.416 (20 °C/20 °C) Density: 1.413 g/cm3 at 20.1 ± 0.1 °C Batch 21ADM93, 98.7% w/w

[Cousin,1993a, M-162115-01-1]

OECD 109 EPA 63-7 EC A3 pyknometer

Specific gravity 1.421 (20 °C/20 °C) Density 1.418 g/cm3 at 20.1 ± 0.1 °C Batch FP1308 98.3% w/w

[Cousin,1993a, M-162115-01-1]

OECD 109 EPA 63-7 EC A3 pyknometer

Melting range: 136 ± 1 °C Batch 21ADM93, 98.7% w/w

[Cousin,1993a, M-162115-01-1]

EPA 63-5 EC A1 Differential

Isoxaflutole

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Parameter Result References Guidelines/ method Calorimetric Analysis

135 ± 1 °C Batch FP1308, 98.3% w/w

[Cousin,1993a, M-162115-01-1]

EPA 63-5 EC A1 Differential Calorimetric Analysis

Thermal stability Thermal decomposition above 160 °C Batch 21ADM93, 98.7% w/w

[Cousin,1993a, M-162115-01-1]

EC A2, Differential Calorimetric Analysis

Thermal decomposition above 160 °C Batch FP1308, 98.3% w/w

[Cousin,1993a, M-162115-01-1]

EC A2, Differential Calorimetric Analysis

Stability in air: Stable for at least 2 weeks at 54 °C. Stable for at least 2 years at ambient temperature Batch 21ADM93, 98.7% w/w

[Cousin, 1995b, M-162149-02-1]

OECD 113, EPA 63-13

Formulations

Isoxaflutole has not been evaluated by JMPS and therefore no FAO specifications for technical and formulated isoxaflutole have been published.

The following formulations with isoxaflutole are commercially available: SL formulations (479 g ai/L), WG formulations (750 g ai/kg), SC formulations (240 g ai/L, 479 g ai/L and 480 g ai/L), SC formulations (44 g ai/L and 240 g ai/L) in combination with a safener cyprosulfamide, SC formulations (50 g ai/L and 225 g ai/L) in combination with a safener cyprosulfamide and a herbicide thiencarbazone-methyl.

Table 1 List of reference compounds used in various study reports

Abbreviation

Trivial and systematic chemical names Other abbreviations used in study reports

Found as or in

parent, IFT MW=359.3

Isoxaflutole, RPA 201772, AE B197278; RPA 591428; 94 BCS-AH21981 IFT; 4-(2-methanesulfonyl-4-trifluoromethylbenzoyl)-5-cyclopropyl isoxazole; 5-cyclopropyl-4-(2-methanesulfonyl-4-trifluoromethylbenzoyl)- isoxazole IUPAC: 5-cyclopropyl-1,2-oxazol-4-yl α,α,α-trifluoro-2-mesyl-p-tolyl ketone CAS: (5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl] methanone CAS No.: 141112-29-0

Glyphosate/HPPD-tolerant soya bean forage and hay (only in post emergent application), wheat hay, sugar cane plants (only after post emergence application). Rat (high dose animals only): urine (< 0.22%) and faeces (< 8.2%). Soil compound

IFT-DKN Isoxaflutolediketonitrile; Rat: major metabolite in urine

CF3

SO2CH3O

NO

Isoxaflutole

1176

Abbreviation


Found as or in

MW = 359.3

RPA 202248, DKN; Diketonitrile metabolite of isoxaflutole; AE 0540092; 14733 BCS-AB59005; 2-cyano-3-cyclopropyl-l-(2-methylsulfonyl-4-trifluoromethylphenyl) propane-1,3-dione; 2-cyclopropylcarbonyl-3-(2-methylsulfonyl-4-trifluoromethylphenyl)-3-oxopropanenitrile; IUPAC: 3-cyclopropyl-2-[2-mesyl-4-(trifluoromethyl)benzoyl]-3-oxopropanenitrile; CAS: α-(cyclopropyl-carbonyl)-2-(methylsulfonyl)-β-oxo-4-trifluoromethyl)- benzenepropanenitrile; CAS No.: 143701-75-1

(28–64%), faeces (21–44%) and liver. Hen: major metabolite in egg yolk, muscle, fat, skin, liver and kidney. Goat: major metabolite in milk, liver, kidney, muscle, renal and omental fat. Maize: minor metabolite in sweet corn, forage, fodder and grain. Glyphosate/HPPD-tolerant soya bean: (major) metabolite in forage, hay and seed. Wheat: minor metabolite in hay and straw. Sugar cane: minor metabolite in whole plant. Poppies: minor metabolite in seed bolls and upper stem and straw. Soil aerobic degradation product Soil photolysis product Rotational crop metabolite: radish leaf, sorghum grain Hydrolysis product Photodegradation product

IFT-BA MW = 268.2

Isoxaflutole benzoic acid IFT Benzoic acid, BA, RPA 203328, IFT-acid benzoic acid metabolite of isoxaflutole AE B197555; 10069 BCS-AB49990 Pyrasulfotole benzoic acid; IUPAC: 2-mesyl-4-trifluoromethylbenzoic acid; CAS: 2-(methylsulfonyl)-4-trifluoromethyl-benzoic acid CAS No.: 142994-06-7

Rat: minor metabolite in urine and faeces (0.58–3.6%). Hen: minor metabolite in muscle and possibly kidney. Maize: major metabolite in forage, sweet corn, forage, fodder and grain. Glyphosate/HPPD-tolerant soya bean: (major) metabolite in forage, hay and seed. Wheat: major metabolite in hay, grain and straw. Sugar cane: major metabolite in whole plant. Poppies: major metabolite in seeds, seed bolls and upper stem and straw. Soil aerobic degradation product Soil photolysis product Rotational crop metabolite: radish leaf, lettuce leaf, sorghum forage, sorghum fodder, sorghum grain, wheat forage, wheat straw, wheat grain Photodegradation product

RPA 205834 MW = 361.3

AE 0692291; 10361 BCS-BY16134; 2-aminomethylene-l-cyclopropyl-3-(2-methylsulfonyl-4-trifluoromethyl-phenyl) propane 1,3-dione; 2-aminomethylene-1-cyclopropyl-3-(2-methylsulfonyl α, α,α-trifluoro-p-tolyl)propane-1,3-dione; IUPAC: 2-aminomethylene-l-cyclopropyl-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione

Rat: minor metabolite in urine (< 2.3%) and faeces (< 1.5%). Hen: minor metabolite detected in yolk. Goat: minor metabolite detected in milk, renal and omental fat. Photodegradation product

CF3

OO SO2CH3

CN

CF3

O SO2CH3

OH

Isoxaflutole

1177

Abbreviation


Found as or in

↕

RPA 207048 MW = 360.3

AE 0893029; 10054 IUPAC: 1-cyclopropyl-2-hydroxymethylene-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione

↕

Rat: minor metabolite in faeces (< 1.9%). Hen: minor metabolite in muscle and fat. Goat: minor metabolite in milk, liver, kidney, muscle, renal and omental fat.

IFT-amide MW= 267.2

Isoxaflutole benzamide IUPAC: 2-mesyl-4-trifluoromethyl benzamide CAS: 2-(methylsulfonyl)-4-(trifluoromethyl) benzamide

Glyphosate/HPPD-tolerant soya bean: (major) metabolite in forage, hay and seed after pre-plant applications. Minor metabolite in post emergent applications (hay and seeds).

RPA 204497 MW = 282.2

methyl-2-mesyl-4-trifluormethylbenzoate

–

RPA 205568 MW=281.2

5-cyclopropyl-1,2-oxazol-4-yl-α,α,α-trifluoro-p-tolyl ketone

Rat: minor metabolite in urine and faeces.

CF3

OO SO2CH3

NH2

CF3

OO SO2CH3

NH

CF3

OO SO2CH3

OH

CF3

OO SO2CH3

O

CF3

O

NH2

SO2CH3

CF3

O SO2CH3

OCH3

Isoxaflutole

1178

Abbreviation


Found as or in

Met 14 MW= 377

IUPAC: (2Z)-3-hydroxy-2-{hydroxy[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methyl}-5-oxohex-2-enenitrile

Photodegradation product

Met 20 MW = 359

IUPAC : (2Z,4Z)-2-{hydroxy[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methylene}-3-oxohex-4-enenitrile

Photodegradation product

METABOLISM AND ENVIRONMENTAL FATE

The Meeting received information on the fate of isoxaflutole in livestock, plant commodities, soil and rotational crops. In all these studies isoxaflutole was uniformly labelled with 14C in the phenyl ring (Figure 1).

Figure 1 Position of [14C]- radiolabel in isoxaflutole

Animal metabolism

The Meeting received information on the fate of isoxaflutole in ruminants (lactating goat) and poultry (laying hens). The metabolism in laboratory animals was summarized and evaluated by the WHO panel of the JMPR in 2013.

Lactating goats

Radiolabelled [U-14C-phenyl]-isoxaflutole was administered orally by gavage to four lactating goats (British Saanen) twice daily for 7 consecutive days at a nominal dose of 1 (goat 001F), 10 (goat 002F and goat 003F) or 50 (goat 004F) mg/kg feed assuming a daily intake of 2 kg dry matter [Lappin, 1995b, M-166744-01-2]. Animals were dosed in the morning and afternoon prior to feeding and after milk and excreta collections. The mean actual concentrations were 1.084, 10.04, 12.98 and 63.92 mg ai/kg dry feed for goat 001F, 002F, 003F and 004F, respectively. In terms of mg ai/animal, the corresponding daily means were 2.00, 20.1, 20.1 and 100 mg ai/animal. Goats were less than 8 years of age. Body weights were 63 to 87 kg on arrival, 64 to 78 kg at the start of acclimatisation and 61 to 73 kg at the end of the study. It is noted that the animals lost weight during the acclimatisation

CF3

O

NO

CF3

SO2CH3OHO OH

CN

O

CF3

SO2CH3OH

CN

CF3

SO2CH3O

NO

*

Isoxaflutole

1179

period. Recovery occurred in three of the four animals. Average daily feed consumption during application was 1.848, 2.00, 1.545, and 1.566 kg/animal/day for goat 001F, 002F, 003F and 004F, respectively. Average daily milk production during application was 1.628, 2.199, 1.638, and 1.745 kg for goat 001F, 002F, 003F and 004F, respectively, and was not affected by the administration of isoxaflutole. The goats were sacrificed ca 23 hours after administration of the last dose. Selected tissues (skeletal muscle, renal and omental fat) and organs (liver and kidney) were removed. In addition the gastrointestinal tract and its contents were analysed in the case of animals 003F and 004F.

The total radioactive residue (TRR) was assessed by LSC or combustion/LSC. Results are shown in Table 2 and Table 3. The total recovered radioactivity amounted to 96.64% TAR in goat 001F, 88.00% TAR in goat 002F, 77.72% TAR in goat 003F and 72.64% TAR in goat 004F. The majority of the radioactivity was recovered from urine (27 to 54% TAR) and faeces (26 to 31% TAR). Levels of radioactivity in milk were not detectable in the low dose goat (001F) and low in the other three animals (0.57, 0.60 and 0.54% TAR in goat 002F, 003F and 004F, respectively). Moderate levels of radioactivity were found in the tissues (including skin), 11.3, 7.49, 9.66 and 8.83% TAR in goat 001F, 002F, 003F and 004F, respectively).

The total radioactive residues (TRR) in tissues and milk were 0.536 to 3.946 mg/kg eq in liver, 0.164–2.123 mg/kg eq in kidney, 0.011 to 0.235 mg/kg eq in omental fat, 0.037 to 0.927 mg/kg eq in muscle, and reaching a maximum of 0.060, 0.095 and 0.350 mg/kg eq in milk for the three highest dose levels (10, 10 and 50 mg ai/kg dry feed), respectively. A steady state was apparent 48 to 96 hours after the first dose for the two low dose levels (1 and 10 mg ai/kg dry feed) with steady state levels of 0.053 mg/kg eq and 0.076 mg/kg eq, respectively. For the high dose group, a steady state was observed by 120 hours after first dosing with a mean concentration of 0.338 mg/kg eq. No separation of the milk in skimmed milk and cream was performed.

Samples were stored at ca -20 °C and analysed within 6 months after sampling. Storage stability was established by a comparison of the nature of radiolabelled residues in urine collected from animal 002F in three HPLC systems over an 18 month period, demonstrating no notable qualitative or quantitative differences in the profiles.

Table 2 Distribution and recovery of administered radioactivity in lactating goats fed with isoxaflutole

Dose 1 mg ai/kg dry feed

10 mg ai/kg dry feed

10 mg ai/kg dry feed 50 mg ai/kg dry feed

%TAR (mg/kg eq)

%TAR (mg/kg eq)

%TAR (mg/kg eq)

%TAR (mg/kg eq)

Urine 54.27 53.90 27.20 27.07 Faeces 31.04 26.04 28.29 29.35 GIT n.a. n.a. 1.642 2.496 Cage wash nd nd 11.77 5.836 Milk nd 0.567 0.599 0.539 Edible tissues and organs (total)

11.34 7.49 7.1 5.3

Omental fat 0.232 (0.011)

0.104 (0.100)

0.187 (0.062)

0.198 (0.235)

Renal fat 0.069 (0.015)

0.042 (0.092)

0.082 (0.069)

0.043 (0.230)

Muscle 7.258 (0.037)

5.397 (0.276)

5.322 (0.263)

4.347 (0.927)

Liver 3.576 (0.536)

1.821 (2.260)

1.400 (2.100)

0.706 (3.946)

Kidney 0.208 (0.164)

0.125 (0.969)

0.085 (0.905)

0.052 (2.123)

Skin n.a. n.a. 0.939 0.990 Total 96.65 88.00 77.52 71.63

nd = not detected; n.a. = not applicable; GIT = gastro intestinal tract

Isoxaflutole

1180

Table 3 Mean total radioactive residue concentration in milk in mg/kg equivalents

Time (hr)

mg/kg eq 1 mg/kg feed 10 mg/kg feed 10 mg/kg feed 50 mg/kg feed

24 nd 0.032 0.045 0.159 48 nd 0.045 0.055 0.260 72 nd 0.048 0.063 0.286 96 nd 0.051 0.095 0.310 120 nd 0.054 0.093 0.350 144 nd 0.059 0.076 0.335 168 nd 0.060 0.074 0.329 174 nd 0.042 0.047 0.267

nd = not detected (≤ 0.002 mg eq/kg)

For further characterisation and identification the homogenates of the tissues of goat 003F and milk of goat 002F (10 mg ai/kg dry feed group) were subjected to further analysis.

Milk was mixed with chloroform and MeOH and partitioned. The aqueous MeOH fraction was removed and partitioned against chloroform. Chloroform fractions were pooled and reduced to low volume. The pellet from the initial extraction partition procedure was extracted with MeOH, which was pooled with the other MeOH fractions and then admixed with the concentrated chloroform extracts. This was subjected to SPE and eluted with MeOH. The aqueous residue was reconstituted with water prior to analysis by HPLC and HPLC-MS.

Kidney and liver homogenate was extracted sequentially with K2HPO4 buffer pH 7.5. Extracts were pooled and mixed with ACN and concentrated. The liver extract was reconstituted in mobile phase prior to HPLC and the kidney extract to HPLC. These initial analyses did not yield the requested results. Hence the extracts were further treated by mixing with ACN and particulate matter and precipitated salts were removed by centrifugation during concentration of the extract using nitrogen convection. The extracts were reconstituted in mobile phase prior to HPLC and HPLC-MS analysis.

Muscle homogenate was extracted sequentially with K2HPO4 pH 7.5 buffer and NH4Ac pH 4.0 buffer. Extracts were pooled and mixed with ACN and concentrated. The extract was mixed with mobile phase and analysed by HPLC. A desalting procedure was undertaken as performed for kidney tissue, but the procedure had little effect on the HPLC chromatography. Fresh tissue sample was extracted in K2HPO4 pH 7.5 buffer. The extract was diluted 40 times with 2% aqueous formic acid and applied to a SPE column. Radioactivity was eluted from the column using 2% formic acid in acetone. Eluent was dried by nitrogen convection and reconstituted in HPLC mobile phase and analysed by HPLC. One minute fractions were collected and analysed by LSC.

A sample of the muscle extract was digested with protease in a similar manner as was done for the solid phase (see below). Further purification was performed by dilution with 2% aqueous formic acid and applying SPE as described above. The acetone eluent was reconstituted in HPLC mobile phase and analysed by HPLC. One minute fractions were collected and analysed by LSC.

Further processing of the solids was conducted by re-suspension in K2HPO4 buffer pH 7.5 with protease, incubation overnight (37 °C), freeze drying and mixing with mobile phase and ACN. The sample was concentrated and cleaned prior to reconstituting the extract with mobile phase and analysis by HPLC and HPLC-MS.

Renal fat was homogenised with hexane and mixed with K2HPO4 buffer pH 7.5. The mixture was centrifuged and organic and aqueous fractions removed. The procedure was repeated twice with the resultant pellet. The hexane fractions were combined, but not further characterized. The aqueous fractions were combined and the excess buffer removed by freeze drying. The resultant residue was mixed with ACN and excess particulate matter removed using centrifugation. The sample was concentrated and cleaned. The resultant residue was reconstituted in mobile phase prior to analysis by HPLC and HPLC-MS.

Isoxaflutole

1181

Omental fat was extracted in the same manner as renal fat. In addition, another extraction method was applied to a second sample, where omental fat was extracted sequentially with ACN (3×), MeOH, and acidified MeOH (2×). The resulting residue was mixed with hexane and partitioned sequentially against ACN, MeOH, acidified MeOH (3×), and water (2×). All extracts (excluding the hexane fraction) where combined, excess solvent removed and cleaned of particulate matter. The remaining aqueous sample was then analysed directly by HPLC.

The nature and identity of the residues in extracts (solvent and/or protease hydrolysates) of milk, kidney, muscle, omental fat and renal fat sampled from animals 002F and 003F were investigated using HPLC and HPLC-MS. Characterisation and/or identification in extracts were based on co-chromatography of reference standards IFT-DKN, IFT-BA, RPA 207048, and RPA 205834.

Results are shown in Table 4. Parent IFT was not found in any of the animal commodities. Three degradation products were identified in the different goat matrices. In milk, muscle, liver, kidney and fat, the metabolite IFT-DKN was the most abundant component of the residues (25–86% TRR or 0.015–1.80 mg/kg eq), followed by the metabolite RPA 207048 (12–26% TRR or 0.009–0.261 mg/kg eq) in all matrices and metabolite RPA 205834 (8.1–18.3% TRR or 0.005–0.011 mg/kg eq) in milk and omental fat only. Several unknown residues were characterized (2.66% TRR to 24.2% TRR or 0.007–0.050 mg/kg eq) in muscle, renal fat, omental fat and milk. Metabolite IFT-BA was not found in any fraction.

The solids remaining after initial extraction in kidney and muscle were treated with protease hydrolysis, resulting in a release of more IFT-DKN and RPA 207048 in muscle (respectively 0.061 mg/kg eq (23.2% TRR) and 0.009 mg/kg eq (3.8% TRR). Part of the radioactivity could not be attributed to any of the known metabolites (0.050 mg/kg eq (5.6% TRR) in kidney and 0.041 mg/kg eq (15.6% TRR) in muscle).

Table 4 Characterisation of residues in goat 002F (milk) and goat 003F (tissues) receiving 10 mg ai/kg dry feed

Tissue TRR mg/kg eq

IFT-DKN mg/kg eq (%TRR)

RPA 207048 mg/kg eq (%TRR)


Unk 1 mg/kg eq (%TRR)



PES mg/kg eq (%TRR)

Total %TRR

Milk 0.060 0.025 (41.7%)

0.009 (15.0%)

0.011 (18.3%)

0.012 (20.0%) a

– – 95.0%

Liver 2.100 1.801 (85.8%)

0.261 (12.4%)

– – – – 98.2%

Kidney 0.905 0.742 (82%)

0.105 (11.6%)

– – – 0.050 (5.57%)

99.2%

Muscle 0.263 0.048 (18.3%) 0.061 b

(23.2%) b

0.024 (9.13%) 0.009 b

(3.42%) b

– 0.007 (2.66%) b

0.010 (3.80%)b

0.041 (15.6%) b,

c

– 76.0%

Omental fat

0.062 0.015 (24.2%)

0.016 (25.8%)

0.005 (8.07%)

0.010 (16.1%)d

0.015 (24.2%)e

– 98.4%

Renal fat 0.069 0.017 (26.6%)

0.013 (18.8%)

0.010 (14.5%)

0.011 (15.9%)f

0.013 (18.8%)e

– 92.8%

TRR: total recovered residue; Unknown 1 in aqueous fraction of omental fat and renal fat and in protease hydrolysate of muscle; Unknowns 2 in hexane fraction of omental fat and renal fat and in protease hydrolysate of muscle; Unknown 3 after protease hydrolysis of muscle solids a Composed of at least six radiolabelled metabolites (highest peak 0.009 mg/kg eq and < 10%TRR). b Released following protease hydrolysis of solids c Composed of at least two radiolabelled metabolites (highest peak 0.011 mg/kg eq, but < 10% TRR) d Composed of at least three radiolabelled metabolites in the aqueous fraction (highest peak 0.008 mg/kg eq, 12.1% TRR). e Partitioned into hexane, not further characterized. Taken into account the low residue levels and the common metabolism between plant and animals, this fraction is not expected to contain any toxicologically relevant residues. f Composed of at least three radiolabelled metabolites in the aqueous fraction (highest peak 0.011 mg/kg eq, 15.9% TRR)

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1182

Laying hens

Ten laying hens (ISA Warren strain) were orally dosed by gavage once daily with [U-14C-phenyl]-isoxaflutole at a nominal dose rate of 1 (group A) and 10 (group B) mg ai/kg dry feed (based on an assumed feed intake of 150 g dry matter) for 14 consecutive days [Lappin, 1995a, M-170844-01-1]. The nominal dose is equivalent to 0.15 mg ai/animal for group A and 1.5 mg ai/animal for group B. Average body weights were 1.78 and 1.82 kg at the start of the study for group A and B, respectively and 1.80 for both groups at necropsy (range 1.67–2.0 kg). The mean actual dose administered, based on the actual mean food consumption of 0.134 kg diet/day, was 1.135 and 11.35 mg ai/kg dry feed for dose group A and B, respectively. The eggs were collected twice daily and excreta in 24 hr intervals. Eggs were kept refrigerated and separated in egg yolks and egg whites prior to radio analysis. Approximately 23 hours after the last dose, the hens were sacrificed and the tissues (fat, muscle and skin) and organs (kidney and liver) were removed. Samples were stored at -20 °C.

The collected samples were analysed by combustion LSC. The total recovery of radioactivity was found to be 117.1% in dose group A. Radioactivity from the excreta and cage wash amounted to 112.4% of the total radioactivity administered. Negligible radioactivity was recovered in egg yolk (0.15% TAR) or tissues (1.7%). Levels of radioactivity were not detected in egg white. The total recovery of radioactivity in dose group B was found to be 92%. The majority of the radioactivity was recovered in the excreta (88.5%), negligible radioactivity was recovered in the egg yolk (0.10%), egg white (0.02%) or tissues (0.20%). Results are shown in Table 5.

Table 5 Distribution and recovery of total administered radioactivity (% TAR) in laying hens dosed with isoxaflutole

Matrix 1 mg ai/kg dry feed (n=5)

10 mg ai/kg dry feed (n=5)

Excreta 112.4 88.5 Cage wash 2.83 3.24 Egg white nd 0.10 Egg yolk 0.15 0.02 Tissues (combined) 1.71 0.20 Muscle < LOD 0.035 mg/kg eq Fat < LOD a 0.028 mg/kg eq Liver 0.845 mg/kg eq 0.935 mg/kg eq Kidney 0.055 mg/kg eq 0.155 mg/kg eq Skin 0.008 mg/kg eq 0.068 mg/kg eq Total 117.1 92.01

nd = not detected (limits of detection were calculated on the basis of the levels of the control samples and were between 0.002 and 0.007 mg/kg eq for all tissues) a except for one animal

In the low dose group (A) the concentrations of radioactivity in egg whites were below the limit of detection, < 0.002–0.003 mg/kg eq, at all-time points, and above the limit of detection in egg yolk (0.002–0.003 mg/kg eq) as from day 4, with maximum mean concentration of 0.028 mg/kg eq on day 10 and a steady state within 7 days after the first dose (ca 0.022 mg/kg eq). In the high dose group (B) the concentration of radioactivity in egg whites reached a maximum of 0.015 mg/kg eq after 10 days of exposure. A steady state (ca 0.010 mg/kg eq) in egg white was reached within 4 days of exposure. The levels of radioactivity in egg yolk were up to 16 times higher, with a maximum of 0.152 mg/kg eq after 12 days of exposure and a steady state concentration of ca 0.137 mg/kg eq within 7 days of exposure.

The highest radioactivity concentrations in edible tissues were found in the liver (0.845 and 0.953 mg/kg eq for dose group A and B, respectively) and kidney (0.055 and 0.155 mg/kg eq, respectively for A and B. Radioactivity concentrations in fat and muscle were only observed in the high (B) dose group, being 0.028 and 0.035 mg/kg eq, respectively. Some radioactivity was found in skin (0.008 and 0.068 mg/kg eq in dose groups A and B, respectively).

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1183

The nature of the radioactive residues was investigated in excreta and tissues of the animals of dose group B (10 mg/kg feed)

A variety of extraction and clean-up methods were employed to analyse residues in the edible tissues and eggs. Subsamples of eggs and homogenized subsamples of tissues and organs (combined samples of five animals from group B) were generally extracted with MeOH (3–4 times), sometimes followed by sequential extraction with acidified MeOH and water (kidney and skin). The aqueous residue was backwashed using hexane and the hexane wash was backwashed with MeOH. The MeOH wash was combined with the aqueous residue and reduced to low volume prior to analysis by HPLC and HPLC-MS. For kidney the aqueous extracts were freeze dried and reconstituted in MeOH and then pooled with the MeOH residue. Excess MeOH was removed and the residue reconstituted in water prior to analysis by HPLC-MS. Following analysis the remaining residue (kidney and liver) was mixed with MeOH and aqueous ACN, the excess solvent removed and reconstituted in water prior to analysis by HPLC.

Egg yolk was extracted subsequently with hexane, MeOH, ACN, acidified MeOH and water. Further extraction using ACN, EtOAc, acidified water and acidified EtOAc did not release any further radioactivity and were therefore not included. Other extracts were combined and excess solvent removed and cleaned up by hexane backwashes and centrifugation to remove participated particulate matter. The resultant residue was reconstituted in mobile phase prior to analysis by HPLC and HPLC-MS.

Egg white was extracted sequentially with ACN, MeOH, EtOAc, acidified MeOH, water and ACN. Quantitative recovery of radioactivity was not achieved following this exhaustive solvent extraction.

Remaining solids of kidney, skin, muscle and egg yolk and white were further subjected to protease digestion (48 hr, 37 ºC) and subsequently to acid hydrolysis (6 M HCl at 95 ºC for 7 days). The remaining pellets were analysed for residual radioactivity by combustion. For skin the only [14C] residue levels in the protease hydrolysate were greater than 0.010 mg/kg eq and for the muscle and egg yolk the levels in the acid hydrolysate were greater than 0.010 mg/kg eq. These fractions were further prepared for analysis by HPLC and HPLC-MS.

All fractions were analysed by LSC. The identification and quantification of metabolites in the various fractions was accomplished by HPLC and LC-MS. Characterisation and/or identification in extracts was based on co-chromatography of reference standards for IFT-DKN, IFT-BA, RPA 207048, and RPA 205834. Identification and characterisation of isoxaflutole related residues is shown in Table 6.

In egg yolk metabolites IFT-DKN (26.3% TRR, 0.036 mg/kg eq) and RPA 205834 (27.7%, 0.038 mg/kg eq) were identified. In the metabolic organs liver and kidney the majority of the radioactivity represented IFT-DKN (93.1% TRR and 73.6% TRR, respectively, equivalent with 0.887 mg/kg eq and 0.114 mg/kg eq). The same applies for the skin (61.8% TRR or 0.042 mg/kg eq, being the sum of the findings in the extractable residue and release after protease and acid hydrolysis). In muscle the main metabolite identified was RPA 207048, 48.5% TRR or 0.017 mg/kg eq (combined results of extractable residue and acid hydrolysate). In fat the main metabolites identified were IFT-DKN (28.6% TRR or 0.008 mg/kg eq) and RPA 207048 (21.4% TRR or 0.006 mg/kg eq).

Samples were stored at ca –20 °C and analysed within 6 months after sampling. Storage stability was established by a comparison of the nature of radiolabelled residues in excreta collected from animal 006F (group A) and 010F (group B) in three HPLC systems over an 18 month period, demonstrating no notable qualitative or quantitative differences in the profiles.

Note: More than 50% of the radioactivity in egg yolk could not be identified (12.4% TRR in solvent extract) and 46% TRR after acid hydrolysis.

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Table 6 Identification and characterisation of isoxaflutole related residues in hens dosed with 10 mg ai/kg dry feed

Tissue TRR mg/kg eq




IFT-BA mg/kg eq (%TRR)

Unk mg/kg eq (%TRR)

Unk mg/kg eq (%TRR)

Unk mg/kg eq (%TRR)

PES mg/kg eq (%TRR)

Total (%TRR)

Liver 0.953 0.887 (93.1%)

– –

– 0.033 a

(3.46%) – – – 0.034

(3.57%) 100.1%

Kidney 0.155 0.114 (73.6%)

– –

– 0.001 b

(0.65%) – 0.025 c,d

(16.1%) 0.003 e

(1.94%) – 92.3%

Muscle 0.035 0.002 e

(5.71%) 0.011 (31.4%) 0.006 e

(17.1%)

– 0.002 e

(5.71%) 0.008 f (22.9%)

0.010 c

(28.6%) 0.005 e, g

(14.3%) – 125.7%

Fat 0.028 0.008 (28.6%)

0.006 (21.4%)

– – 0.012 h

(42.9%) – – – 92.9%

Skin 0.068 0.025 (36.8%) 0.017 j (25%)

– – – 0.011 i (16.2%)

0.002 c,k

(2.94%)c,k – – 80.9%

Egg yolk

0.137 0.036 (26.3%) 0.023 e

(16.8%)

– 0.038 l (27.7%)

– 0.017 m

(12.4%) – 0.040 n

(29.2%) – 112.42%

Egg white

0.010 – – – – 0.005 (50%)

0.002 (20%)c

0.002 e

(20%) – 90%

a Contains IFT-BA indicated by HPLC-MS. b Resolved in only one HPLC system and tentatively identified as IFT-BA by HPLC-MS. c Released after protease digestion d Composed of at least three unknown radiolabelled metabolites, with the highest peak being 0.009 mg/kg eq. e Released after acid hydrolysis f Composed of at least three unknown radiolabelled metabolites in approximate ratio 4:3:1 ( highest peak 0.004 mg/kg eq). g Composed of at least two unknown radiolabelled metabolites in approximate ratio 3:2 (highest peak 0.003 mg/kg eq), h Composed of at least two unknown radiolabelled metabolites in approximate ratio of 1:1 (highest peak 0.006 mg/kg eq) i Composed of at least three radiolabelled metabolites in the approximate ration of 5:4:2 (highest peak 0.005 mg/kg eq). j 0.012 mg/kg eq (17.7% TRR) was released after protease digestion and 0.005 mg/kg eq (7.35%TRR) was released after acid hydrolysis. k Composed of at least one metabolite. l Identification based on similar chromatographic properties in a single HPLC system. m Composed of at least two radiolabelled metabolites in the approximate ratio of 5:1 (highest peak 0.014 mg/kg eq) n Released following acid hydrolysis and composed of at least three radiolabelled metabolites (0.004–0.011–0.025 mg/kg eq). No radiolabelled metabolites were released following protease treatment.

Overview metabolic pathway in livestock

Isoxaflutole was transformed to a number of metabolites after administration to ruminants and poultry. The proposed metabolic pathway of isoxaflutole in livestock involves the opening of the oxazole ring and the formation of the diketo-nitrile derivative (IFT-DKN) or the diketo-amine derivative (RPA 205834). Further degradation occurs through deamination to form the diketo-hydroxy derivative (RPA 207048) or through further cleavage to form the benzoic acid derivative (IFT-BA). Further metabolism involves conjugation of IFT-DKN, RPA 207048 and/or IFT-BA with proteins or acid cleavable compounds. The pathway is shown schematically in Figure 2.

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1185

Figure 2 Proposed metabolic pathway of isoxaflutole in livestock

Plant metabolism

The Meeting received information on the uptake and metabolic fate of isoxaflutole in cereals (maize and wheat), pulses/oilseeds (soya beans, poppy seeds) and sugar cane. Further the Meeting received information on the effect of the addition of the safener cyprosulfamide on isoxaflutole related residues. The test item used in all studies was [U-14C-phenyl]-isoxaflutole (see Figure 1).

Study 1

The metabolic fate and distribution of isoxaflutole was studied outdoors in maize (Zea mays L) after either a pre-plant incorporated (PPI) or pre-emergence (PRE) application [Hampton and Pettaway, 1995, M-162883-01-1]. The total amount applied was 0.209 or 0.657 kg ai/ha for the PPI application and 0.227 or 1.075 kg/ha for the PRE application. The test item used was [U-14C-phenyl]-isoxaflutole. The soil characteristics were: sandy loam, pH 5.3, 1.43% om, 2.94 meq/100 g CEC. The active ingredient was applied on day 1 to the soil either before planting (PPI) or after planting (PRE) as dosing solution using pipettes and applying it in a criss-cross pattern to the soil. For the PPI treatment

CF3

SO2CH3O

NO

CF3

OO SO2CH3

CN

CF3

O SO2CH3

OH

CF3

OO SO2CH3

NH2

CF3

OO SO2CH3

OH

Isoxaflutole (RPA201772)

IFT-DKN (RPA 202248)hen: major metabolite in egg yolk, muscle, fat, skin, liver and kidney.goat: major metabolite in milk, liver, kidney, muscle and renal and omental fat.

RPA 207048hen: major metabolite in muscle and fatgoat: minor metabolite in milk, liver, kidney,muscle, renal and omental fat

IFT-BA (RPA 203328)hen: minor metabolite in muscle(and possibly kidney)

hydrolysis

RPA 205834hen: major metabolite in egg yolkgoat: minor metabolite in milk, renal and omental fat

IFT-DKN (RPA 202248)-conjugates

IFT-BA (RPA 203328)-conjugatesRPA 207048-conjugates

Isoxaflutole

1186

the application was subsequently incorporated in the soil with a rake to the depth of approximately 2.5 cm, before planting. Plants were harvested at immature and mature stages (one plant of the container for the forage harvest and one for fodder and grain harvest). Forage was harvested at 41 DAP/DAT (BBCH growth stage not reported, plants were 51–61 cm tall) and comprised of the combined leaves and stems. The mature plants were harvested at 122 DAP/DAT (leaves and ears) and at 138 DAP/DAT (remaining stems). Fodder samples comprised the leaves, stems, husks, shanks, silks and cob. Grain samples consisted of the shelled grain only. The frozen samples were stored at -20 °C.

Homogenised samples were analysed by combustion LSC. Samples with TRRs greater than 0.01 mg/kg eq were extracted sequentially with hexane/EtOAc (9:1 v/v); ACN; H2O (pH 5.5); and ACN/0.2M HCl (1:1 v/v). Remaining solids in forage and fodder were digested enzymatically by cellulase (6–72 hrs for forage and 24–120 hrs for fodder, 37 ºC, pH 4.8). The digests were acidified (pH < 2) and partitioned against EtOAc. Extracts and remaining solids were quantified by LSC or combustion LSC. All aqueous extracts were acidified and partitioned against EtOAc. All organic extracts and EtOAc layers were concentrated. The concentrated extracts were analysed by HPLC and TLC using reference substances for IFT, IFT-DKN and IFT-BA. The identity of the parent and metabolites was confirmed by HPLC-MS-MS.

The total radioactive residues based on combustion of maize forage, grain and fodder are shown in Table 7. Exaggerated dose rates exhibited greater phytotoxicity. The exaggerated rate treatments were not further taken into account for determining extractability and identification of metabolites.

Table 7 Mean radioactive residues in maize forage, fodder, and grain after normal and exaggerated doses

Matrix Treatment - harvest Use rate kg ai/ha

TRR mg/kg eq

Forage PPI–DAT 41 0 0.000 0.209 0.198 0.657 0.800 PRE–DAT 41 0 0.000 0.227 0.228 1.075 0.491 Grain PPI–DAT 122 0 0.000 0.209 0.044 0.657 0.152 PRE–DAT 122 0 0.000 0.227 0.039 1.075 0.125 Fodder PPI–DAT 122/138 0 0.000 0.209 0.149 0.657 0.661 PRE–DAT 122/138 0 0.000 0.227 0.120 1.075 0.528

The calculated TRR values were used as 100% TRR for all further calculations. Extractability ranged from 81.3% in fodder to 102.1% TRR in forage. Enzymatic digests of forage and fodder contributed to another 4.0% and 6.5% TRR. Remaining solids in forage, fodder and grain were on average 1.5%, 10.7% and 6.5% TRR. The total accountability ranged from 90.9% to 103.3% TRR. The results are shown in Table 8 and Table 9. Approximately 70–80% of the TRR was identified as IFT-DKN and IFT-BA. Calculations showed an identification rate of 64–89% of the TRR comprising of metabolite IFT-BA, typically greater than 90% of the extracted radioactive residue, representing 64–89% of the TRR. Small amounts of IFT-DKN were found in enzyme treated fractions of forage (ca. 0.5% TRR) and in the ACN fraction of grain (7.5% TRR). The parent IFT was not detected in any maize RAC.

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1187

Storage stability of the stored treated maize forage, fodder and grain was tested at 0, 96 and 253 days. HPLC profiles were nearly identical to the spiking solution. Some breakdown of the parent compound to IFT-DKN occurred (up to 27%), but is considered not critically relevant for the study, as the parent was not found in the forage samples and IFT-DKN is relatively stable in storage.

Table 8 Extractability of radioactivity in maize treated pre-plant incorporated (PPI) or pre-emergence (PRE) soil applications at normal field use rate

RAC TRR a Readily extracted

b Enzyme released Total extracted c PES Account

ability mg/kg eq

mg/kg eq d

% TRR mg/kg eq d

% TRR mg/kg eq d

% TRR mg/kg eq d

% TRR % TRR

Forage PPI 0.198 0.180 91.1 0.010 5.1 0.190 96.2 0.003 1.6 97.9 PRE 0.228 0.226 99.1 0.007 3.0 0.233 102.1 0.003 1.3 103.3 Fodder PPI 0.149 0.111 74.8 0.010 6.5 0.121 81.3 0.014 9.7 90.9 PRE 0.120 0.095 78.9 0.008 6.5 0.102 85.4 0.014 11.8 97.2 Grain PPI 0.044 0.038 87.4 n.a. n.a. 0.038 87.4 0.003 7.5 94.9 PRE 0.039 0.033 83.8 n.a. n.a. 0.033 83.8 0.004 10.4 94.2

n.a.= not applicable; n.r. = not reported a TRR = based on initial TRR determined by combustion. b 4 extraction steps combined (HEX/EA, ACN, water and acid extraction); c Total extracted is the sum of the readily extracted and enzyme released radioactivity d calculated by the present reviewer based on %TRR and total TRR values for combustion

Table 9 Characterisation of isoxaflutole related residues from maize forage, fodder and grain

PPI/PRE Extract Total IFT mg/kg eq (%TRR)



Forage PPI 0.209 kg ai/ha DAT 41

TRR 0.190 nd 0.001 (0.53% TRR)

0.138 (72.6%TRR)

HEX/EA 0.003 (1.4%TRR)

– n.a. –

ACN 0.090 (45%TRR)

nd trace 0.082 (43.2%TRR)

water a,b 0.049 (27%TRR)


ACID a 0.026 (14%TRR)

– n.a. –

ENZ a 0.010 (5.1%TRR)

– 0.001 (0.53%TRR)

0.002 (1.1%TRR)

Total extracted d

0.178 (94%TRR)

0.001 (conjugate) (0.53% TRR)

0.136 (free) (71.6% TRR) 0.002 (conjugate) (1.1% TRR)

Total identified d

73.1% TRR

PRE 0.227 kg ai/ha DAT 41

TRR 0.204 0.000 0.001 (0.49% TRR)

0.185 (89.2%TRR)

HEX/EA 0.003 (1.4%TRR)

ACN 0.144 (69%TRR)



nd trace 0.045 (22.1%TRR) a, b

ACID a,b 0.012 (5.8%TRR)

– – –

ENZ a 0.006 – 0.001 0.003

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1188




(3.0%TRR) (0.49%TRR) (1.5%TRR) Total

extracted d 0.205 (100.5% TRR)

– 0.001 (conjugate) (0.49% TRR)

0.182 (free) (89.3% TRR) 0.003 (conjugate) (1.5% TRR)

Total identified d

89.7%TRR

Fodder PPI 0.209 kg ai/ha DAT 122/138

TRR 0.160 nd trace 0.109 (68%TRR)

HEX/EA 0.003 (1.71%TRR)

– n.a. –

ACN 0.028 (18%TRR)



nd nd 0.079 (49.4%TRR)a, b

ACID a,b 0.021 (13%TRR)

– – –a, b

ENZ a 0.010 (6.5%TTR)

nd trace 0.002 (1.25% TRR)

Total extracted d

0.119 (80%TRR)

– – 0.107 (free) (66.9% TRR) 0.002 (conjugate) (1.25% TRR)

Total identified d

68% TRR

PRE 0.227 kg ai/ha DAT 122/138

TRR 0.113 nd trace 0.072 (64%TRR)

HEX/EA 0.002 (1.5%TRR)

– n.a. –

ACN 0.023 (20%TRR)

nd nd 0.023 (20.4%TRR)


nd trace 0.046 (40.7%TRR)a, b

ACID a,b 0.017 (14%TRR)

– – –a, b

ENZ a 0.007 (6.5%TRR)

nd trace 0.003 (2.7%TRR) conjugate

Total extracted d

0.094 (83%TRR)

– – 0.069 (free) (61.1%TRR) 0.003 (conjugate) (2.7% TRR)

Total identified d

64%TRR

Grain PPI 0.209 kg ai/ha DAT 122

TRR 0.053 nd 0.004 (7.5% TRR)

0.035 (66% TRR)

HEX/EA 0.000 – n.a. – can c 0.032

(61%TRR) nd 0.004

(7.5%TRR) 0.035 (66%TRR)c

water c 0.010 (19%TRR)

– – –c

ACID c 0.004 (6.9%TRR)

– – –c

ENZ n.a. Total

extracted d 0.046 (87%TRR)

0.035 (free) (66% TRR)

Total 73.5%TRR

Isoxaflutole

1189




identified d

PRE 0.227 kg ai/ha DAT 122

TRR 0.043 nd trace 0.029 (67%TRR)

HEX/EA 0.000 – – – ACN c 0.025

(59%TRR) nd trace 0.029

(67%TRR) water 0.007

(18%TRR) – – –c

ACID c 0.003 (7%TRR)

– – –c

ENZ a n.a. – – – Total

extracted d 0.035 (82%TRR)

– – 0.029 (free) (67% TRR)

Total identified d

67%TRR

n.a. = not analysed; nd = not detected (peaks of 200 DPM could still be detected) a Represents the results of the organic layer from partitioning with EtOAc, since the results from the aqueous layer were either not analysed or no radioactivity was observed. b The H2O organic and acid organic extracts were combined prior to analysis due to insufficient radioactivity. c ACN, water- and ACID-organic extracts were combined prior to analyses due to low levels of radio activity. d Total extracted and total identified was calculated by the reviewer, based on reported values for individual extracts and individual compounds respectively. The % TRR for metabolites was calculated by the reviewer, based on mg/kg eq values.

Study 2

The metabolic fate and distribution of isoxaflutole was studied outdoors in maize (N7070 BT) after a post emergence application in combination with safener cyprosulfamide (equal weight fractions). Maize plants were treated with an SC formulation of [U-14C-phenyl]-isoxaflutole at an application rate of 0.211 kg ai/ha [Meyer and Ripperger, 2006, M-268739-01-2]. The soil characteristics were: sandy loam, pH 6.4, 1.7% om, 9.9 meq/100 g CEC. The active ingredient was applied 13 DAP at growth stage V2 (BBCH growth stage not reported) by hand-held pump sprayer. Plants were harvested for forage and sweet corn (kernels and cob with husks removed) on DAT 75 and for fodder and grain on DAT 106. For sweet corn and grain the ears of all the plants were combined. All samples were homogenized and stored frozen at –20 °C (for 4–10 days until extraction and for 4–33 days until analysis).

Aliquots of homogenised samples were combusted to determine the total radioactive residues (TRR) levels. Samples with TRRs greater than 0.01 ppm were extracted sequentially with ACN (3×), ACN/water (4:1) and ACN/water (1:1). Filtered extracts were combined. Extracts and solids were analysed by (combustion) LSC. After a clean-up through C18 SPE, the eluate was concentrated and adjusted to pH 2 with HCl and then partitioned with EtOAc. The EtOAc layer was concentrated and partitioned between hexane and MeOH/water (9:1). In case of forage and sweet corn, this layer was further fractionated by C18 solid-phase extraction. For forage and fodder, the aqueous fractions arising from the EtOAc partitioning were treated with 1 M NaOH (17 hours, 50 ºC) and subsequently partitioned with DCM at both basic and acidic pH. The solids remaining after ACN/water extractions of fodder were subjected subsequently to ASE (Accelerated Solvent Extraction) with 0.5 N H2SO4/ACN (1:1) and aq NH4OH/ACN (1:1). The extracts were sampled for LSC and the solids were sampled for drying and combustion. Identification and quantification in those extracts with sufficient radioactivity was performed by reverse-phase HPLC and confirmation by HPLC-MS-MS. Reference substances IFT, IFT-DKN, IFT-BA and RPA 205834 were used.

TRR (sum of extracts and solids) in forage accounted for 0.134 mg/kg eq, in sweet corn 0.010, fodder 0.10 and grain 0.015 mg/kg eq. TRR in the extracts ranged from 77.3 (grain)–96.3

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1190

(kernels + cobs with husks removed (K + CWHR)) % TRR (see Table 10). The TRR in the solids ranged from 3.7 (K + CWHR) to 22.7 (grain) %TRR, but were all < 0.01 mg/kg eq. Identification results are shown in Table 11. The residues in all the RACs were identified primarily as metabolite IFT-BA (61–67% TRR). A second metabolite, IFT-DKN, was found in the fractions of sweet corn, maize fodder and maize grain, accounting for the remaining identified radioactivity (< 10% TRR and ≤ 0.005 mg/kg eq). Parent IFT was not found.

Storage stability was not addressed as the samples were analysed within 33 days.

Table 10 Extractability of radioactivity in maize after treatment with 0.211 kg ai/ha

Matrix DAT TRR ACN and water extracts Solids (mg/kg eq) (% TRR) (mg/kg eq) (% TRR) (mg/kg eq)

Forage 75 days 0.081 92.9 0.075 7.1 0.006 0.156 nd nd nd nd 0.164 nd nd nd nd Mean 0.134 Sweet corn (K+CWHR)

75 days 0.010 96.63 0.009 3.7 < 0.001

Fodder 106 days 0.120 87.9 0.106 12.1 0.015 0.101 nd nd nd nd 0.078 nd nd nd nd Mean 0.100 Grain 106 days 0.015 77.3 0.012 22.7 0.004

nd = not determined

Table 11 Characterisation and identification of radioactive residues in maize (0.211 kg ai/ha)

Compound Forage (75 DAT) Sweet corn (75 DAT) Fodder (106 DAT) Grain (106 DAT)

%TRR mg/kg eq %TRR mg/kg eq

%TRR mg/kg eq

%TRR mg/kg eq

TRR 0.081 0.010 0.100 0.015

IFT – – – – – – – –

IFT-BA 67.2 0.056 60.9 0.005 63.3 0.076 63.0 0.010

IFT-BA in EtOAc extract –51.8 –0.043 60.9 0.005 –59.2 –0.071 –63.0 –0.010

IFT-BA organosoluble after base hydrolysis

–15.4 –0.013 – – –4.1 –0.005 – –

IFT-DKN a – – 6.5 < 0.001 4.0 0.005 9.8 0.001 Total identified 67.2 0.056 67.4 0.006 67.3 0.081 72.8 0.011 Total characterized b 25.6 0.020 28.7 0.003 20.6 0.024 4.5 < 0.001 Other components a –3.0 –0.002 – – – 11.8 – 0.014 – – Aqueous soluble after base hydrolysis

–15.1 –0.012 –25.6 c –0.002 c –7.7 –0.009 –4.0 –< 0.001

Total extractable 92.9 0.075 96.3 0.009 87.9 0.106 77.3 0.012 PES d 7.1 0.006 3.7 < 0.001 12.1e 0.015e 22.7 0.004 Accountability f 86.1 114.5 114.8 113.2

– = not detected (limit of detection was defined as the background radioactivity (25 DPM for LSC measurements). This was subtracted from each sample. a In EtOAc extract b Includes other components observed in HPLC, hexane, SPE fractions not analysed by HPLC, and water soluble (after base hydrolysis where applicable) c Water soluble 6.1% (< 0.001 mg/kg eq), Methanol soluble 14.3% (0.001 mg/kg eq), Other soluble 5.3% (< 0.001 mg/kg eq). d Residues remaining after exhaustive extractions. e Fibre further extracted with acid (4.3%), and base (1.5%). Remaining PES 6.3% (0.008 mg/kg eq) f Accountability = (TRR extracts + TRR solids)/(TRRs from combustion analysis) × 100.

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Study 3

The metabolic fate and distribution of isoxaflutole was studied under field conditions in wheat (Triticum aestivum) [Unsworth and Clarke, 2000, M-211481-01-1]. [U-14C-phenyl]-isoxaflutole was applied as a solution to immature plants (Zadoks growth stage 30, i.e. early stem elongation, pseudo stem erect - third node on the main stem) at a field rate of 0.055 kg ai/ha or at an exaggerated rate of 0.105 kg ai/ha. Only the results of the intended field rate were provided. The soil characteristics were: loam, pH 7.4, 3.3% om, 15.1 meq/100 g CEC. Plants were harvested at interim (hay) at growth stage Z58-Z73 (Zadoks) at DAT 41 or at final harvest at DAT 93 for grain and straw. All samples were homogenised and stored frozen at –20 °C. Plant parts were weighed and homogenised. TRRs were determined by combustion.

The total radioactive residues (TRR) ranged from 0.058–0.172 mg/kg in hay, grain and straw (see Table 12). Samples were extracted by maceration with ACN and ACN/water. A total of 92.4% TRR, 96.5%TRR and 86.0% TRR were extracted from hay, grain and straw, respectively. For wheat straw, solids were soxhlet extracted with DCM/glacial acetic acid (4 hr) and an additional 3.0% TRR could be released. Following concentration, the combined extracts were subjected to quantitative and qualitative analyses by reverse-phase HPLC with radiodetection. Metabolite identification was confirmed by HPLC-MS-MS. Reference substances IFT, IFT-DKN, and IFT-BA were used.

The major metabolite in hay, grain and straw is IFT-BA, accounting for 65%TRR (0.112 mg/kg eq), 95.8%TTR (0.055 mg/kg eq) and 79.1% TRR (0.084 mg/kg eq), respectively. In hay and grain also metabolite IFT-DKN was found; representing 20.9%TRR (0.036 mg/kg eq) and 9.9%TRR (0.011 mg/kg eq), respectively. IFT was only found in hay (interim harvest), accounting for 6.5%TRR (0.011 mg/kg eq). Parent IFT was not found in grain and straw. A small fraction of the metabolites in straw might be attributed to conjugates, because 3% TRR was released through acid reflux. However, since the primary and refluxed extracts were mixed, this fraction cannot be indicated.

Storage stability was not addressed as the samples were analysed within 6 months (< 90 days) and the plant parts were stored frozen (≤ -20 °C) prior to extraction and refrigerated (4 °C) between handling steps).

Table 12 Distribution of isoxaflutole related residues in wheat matrices following treatment with 0.055 kg ai/ha

Hay—DAT 41 Grain—DAT 93 Straw—DAT 939 TRR (mg/kg eq) 0.172 0.058 0.107 %TRR mg/kg eq %TRR mg/kg eq %TRR mg/kg eq IFT 6.5 0.011 – – – – IFT-BA 65.0 0.112 95.8 0.055 79.1 0.084 IFT-DKN 20.9 0.036 – – 9.9 0.011 Unknowns – – 0.7 < 0.001 – – Solids 7.6 0.013 3.5 0.002 11.0 0.012

– = not detected (< 0.001 mg/kg)

Study 4

The metabolic fate and distribution of isoxaflutole were studied under greenhouse conditions in HPPD (p-hydroxyphenyl pyruvatedioxygenase) tolerant soya bean (FG72 Glytol soya bean) for pre-plant and foliar applications [Nguyen, 2010, M-368555-01-1]. [U-14C-phenyl]-isoxaflutole was applied via hand-held sprayer as an SC formulation at an application rate of 0.330 kg ai/ha either directly to the soils prior to planting or applied uniformly to the soya bean plants at the full flowering stage (BBCH 65 or 57 DAP). The soil characteristics were: silty clay loam, pH 7.1, 1.7% om, 21.5 meq/100 g CEC. Plants were harvested for forage (BBCH 75), hay (BBCH 99) and seed (BBCH 99). All samples were homogenised and stored frozen at ≤ –20 °C prior to extraction.

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Aliquots of homogenised samples were combusted to determine the total radioactive residues (TRR) levels. Samples were extracted several times with ACN/water (4:1) and filtered. The combined extracts were cleaned up with SPE. The remaining hay solids and soya bean seed solids were extracted with MeOH (reflux overnight) and subsequently treated with 1 M NaOH overnight at room temperature. Extracts and solids were analysed by (combustion) LSC. Identification and quantification of residues was accomplished by using reverse phase HPLC and confirmation by HPLC-MS-MS. Reference substances IFT, IFT-DKN, IFT-BA were used.

The total radioactive residues (TRR) in the RACs ranged from 0.149–0.268 mg/kg eq in pre plant RACs and from 0.259–13.128 mg/kg eq in post emergence application samples (Table 13 and Table 14).

The residues in the RACs of the pre-plant treated soya beans (Table 13) were identified primarily as metabolites IFT-amide (8–53%TRR), IFT-BA (27–66%TRR) and IFT-DKN (13–17%TRR). In hay an additional unidentified metabolite was detected (9%TRR (0.042 mg/kg eq)). Parent IFT was not found. A total of 91–93%TRR was identified.

Table 13 Distribution of metabolites in HPPD tolerant soya bean matrices following pre-plant treatment with 0.331 kg ai/ha

Forage (74 DAT) Hay (189 DAT) Seed (189 DAT) TRR (mg/kg eq) a 0.268 0.492 0.149 Metabolite/fraction %TRR mg/kg eq %TRR mg/kg eq %TRR mg/kg eq ACN/water extract 93 0.270 91 0.448 92 0.144 IFT – – – – – – IFT amide 53 0.154 13 0.062 8 0.013 IFT-BA 27 0.048 56 0.278 66 0.105 IFT-DKN 13 0.038 13 0.066 17 0.027 unknown (H3) – – 9 0.042 – – MeOH reflux fraction n.a. n.a. 5 0.024 2 0.003 1 M NaOH fraction n.a. n.a. 3 0.013 1 0.002 PES 7 0.021 2 0.008 5 0.008 Accountability b 100 0.291 101 0.493 100 0.157

n.a. = not applicable; n.r. = not reported a TRR determined by combustion b Determined by taking the sum of the extracted plus remaining solids as percentage of the TRR determined by combustion.

The residues in the forage samples of the post-emergence treated soya beans (Table 14) were identified primarily as IFT (72%TRR, 7.757 mg/kg eq), and metabolites IFT-DKN (18%TRR, 1.943 mg/kg eq), IFT-BA (6%TRR, 0.627 mg/kg eq) and an unidentified metabolite PE-F4 (3%TRR, 0.352 mg/kg eq). The residues in the hay samples of the post-emergence treated soya beans were identified primarily as metabolites IFT-BA (38%TRR, 0.608 mg/kg eq), IFT-DKN (21%TRR, 0.334 mg/kg eq), and IFT (25%TRR, 0.411 mg/kg eq) and IFT-amide (3%TRR, 0.055 mg/kg eq). In seed samples metabolites IFT-BA (62%TRR, 0.160 mg/kg eq), IFT-DKN (24%TRR, 0.0.061 mg/kg eq) and IFT-amide (8%TRR, 0.020 mg/kg eq) were identified. Parent IFT was not found.

Storage stability was not addressed as the samples were stored frozen immediately and analysed within 33 days.

Table 14 Distribution of metabolites in HPPD tolerant soya bean matrices following foliar treatment with 0.331 kg ai/ha

Forage (17 DAT) Hay (132 DAT) Seed (132 DAT) TRR (mg/kg eq) 13.128 1.775 0.259 Metabolite/fraction %TRR mg/kg eq %TRR mg/kg eq %TRR mg/kg eq ACN/water extract 100 10.679 92 1.491 94 0.242 IFT 72 7.757 25 0.411 – – unknown (H1) – – 2 0.026 – –

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Forage (17 DAT) Hay (132 DAT) Seed (132 DAT) TRR (mg/kg eq) 13.128 1.775 0.259 Metabolite/fraction %TRR mg/kg eq %TRR mg/kg eq %TRR mg/kg eq IFT amide – – 3 0.055 8 0.020 IFT-BA 6 0.627 38 0.608 62 0.160 IFT-DKN 18 1.943 21 0.334 24 0.061 unknown (F4) 3 0.352 – – – – unknown (H6) – – 3 0.056 – – MeOH reflux fraction n.a. n.a. 4 0.059 2 0.004 1 M NaOH fraction n.a. n.a. 2 0.030 1 0.003 PES 0.50 0.054 2 0.031 3 0.008 Accountability 82 10.73 91 1.61 99 0.257

n.a. = not applicable;

Study 5

The metabolic fate and distribution of isoxaflutole was studied under field conditions in poppies (Papaver somniferum, cultivar Mieszko) after a pre-emergence spray application, 3 DAS [Klempner, 2009, M-360799-01-1]. [U-14C-phenyl]-isoxaflutole was applied as an SC formulation in combination with a non-radiolabelled safener cyprosulfamide via hand-held sprayer at an actual application rate of 0.108 kg ai/ha directly to bare soil. The soil characteristics were: sandy loam, pH 6.9, 2.12% om, 8.1 meq/100 g CEC. Poppy seeds, seed bolls with upper stem and poppy straw were harvested at maturity (BBCH stage 89–92) at DAT 110. All samples were homogenised and stored frozen at –18 °C.

Aliquots of homogenised samples were combusted and radio assayed by LSC to estimate the total radioactive residues (TRR) levels. The total radioactive residues (TRR) in the RACs were 0.056, 0.779 and 0.725 mg/kg eq in seeds, seed bolls & upper stem, and straw, respectively (see Table 15).

Samples were extracted three times with ACN/water (8:2, v/v) and filtered. The solids were dried and analysed for radioactive residues by combustion and LSC. The extracts were radio assayed (LSC). The combined extracted were cleaned up with SPE. The eluate was collected and analysed by LSC. Less polar fractions were eluted with MeOH/tetrahydrofurane (1/1, v/v) and analysed by LSC.

The metabolic profiles of all extracts were analysed by radio-HPLC using reverse phase HPLC. Identification was mainly achieved by isolation of the metabolites from poopy extract by HPLC, followed by HPLC-MS and by HPLC and/or co-chromatography with reference substances IFT, IFT-DKN, IFT-BA and RPA 205834.

A total of 91.5–97.8% of the TRR was extracted. The residues in the RACs of the pre-plant treated soya beans were identified primarily as metabolite IFT-BA, being 6%TRR (0.037 mg eq/kg) in poppy seed, 94.3%TRR (0.734 mg/kg eq) in seed bolls & upper stem, and 88.7%TRR (0.643 mg eq/kg) in poppy straw). In seed bolls, upper stem and poppy straw minor amounts of AE 202248 were found (2.1%TRR and 3.6%TRR, respectively). This metabolite was not detected in poppy seed. In addition four minor, unidentified metabolites were detected in poppy seeds, two of them in seed bolls & upper stem, and one in poppy straw (range 1.0–5.4%TRR, 0.001–0.029 mg eq/kg). As they accounted for < 10% TRR, further analysis of these metabolites was not performed. Parent IFT was not found. A total of 66–96.4%TRR was identified.

Storage stability was not addressed as the samples were extracted and analysed within 14 days and 16 days (seed bolls with upper stem and poppy seeds, respectively) and 12 weeks (straw).

Table 15 Characterisation of residues of poppy matrices following pre-emergence treatment with 0.108 kg ai/ha

Poppy seeds harvest DAT 110

Seed bolls & upper stem harvest DAT 110

Poppy straw harvest DAT 110

TRR (mg/kg eq) 0.056 0.779 0.725 %TRR mg/kg eq %TRR mg/kg eq %TRR mg/kg eq

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Poppy seeds harvest DAT 110

Seed bolls & upper stem harvest DAT 110

Poppy straw harvest DAT 110

TRR (mg/kg eq) 0.056 0.779 0.725 %TRR mg/kg eq %TRR mg/kg eq %TRR mg/kg eq IFT – – – – – – IFT-BA 66 0.037 94.3 0.734 88.7 0.643 IFT-DKN – – 2.1 0.016 3.6 0.026 Subtotal identified 66.0 0.037 96.4 0.751 92.3 0.670 Unknown 1 5.4 0.003 1.0 0.008 4.0 0.029 Unknown 2 2.5 0.001 – – – Unknown 3 2.7 0.002 0.4 0.003 – – Unknown 4 7.1 0.004 – – – – Subtotal characterized 17.8 0.010 1.4 0.011 4.0 0.029 Extracts not analysed 7.8 0.004 – – 0.5 0.003 Total extractable 91.5 0.051 97.8 0.762 96.8 0.702 PES 8.5 0.005 2.2 0.017 3.2 0.023 Accountability 100 0.056 100 0.779 100 0.725

– = not detected;

Study 6

The metabolic fate and distribution of isoxaflutole was studied under field conditions in sugar cane (variety SP 79-1011) [Unsworth, 1999, M-211498-01-1]. [U-14C-phenyl]-isoxaflutole was applied as a solution either to soil just after sugar cane planting (pre-emergence) or on emerged sugar cane (foliar, 47 days after planting). The actual field rates were 0.210 kg ai/ha and 0.133 kg ai/ha, respectively, for pre-emergence and post-emergence treatments. The soil characteristics were: sandy clay, pH 6.2, 2.2% om, 8.1 meq/100 g CEC. Plants were harvested at DAT 81, 95 and 365 for pre-emergence application and at DAT 40, 95 and 365 for post emergence application. Whole plants were bagged and stored frozen until processing, except for the final harvest samples, where the leaves were separated from the cane. Plant parts were weighed and homogenized. All samples were stored frozen prior to homogenisation.

TRRs were determined by combustion and quantified by LSC. Results are shown in Table 16. Total radioactive residues in the DAT 365 samples were < 0.0001 mg/kg eq and in the DAT 95 samples after post emergence application 0.0065 mg/kg eq and therefore not further extracted. Only the DAT 40 (post-emergence), DAT 81 (pre-emergence) and DAT 95 (pre-emergence) samples were extracted. Extraction with ACN released 65.88%, 78.69% and 84% TRR, respectively in these samples. Remaining solids were sequentially soxhlet extracted with ACN (all samples) or ACN/water (80:20 vv, DAT 40 and DAT81). The solids remaining in the DAT 40 samples were extracted further with ACN/water (50:50, vv), 0.1 M HCl, 0.1 M ammonia, 0.1 M HCl at 60 ºC, 0.1 M ammonia at 60 ºC, reflux in 0.1 M HCl, reflux in 0.1 M ammonia. Extracts and solids were analysed by (combustion) LSC. A total of 90.4% TRR, 95.7%TRR and 93.5% TRR could be extracted in the DAT 40, DAT 81 and DAT 95 samples, respectively.

Following concentration, the combined extracts were subjected to quantitative and qualitative analyses by reverse-phase HPLC. Parent and metabolite identification was confirmed by HPLC-MS. Certified reference standards for IFT, IFT-DKN and IFT-BA were used.

The major metabolite in sugar cane is IFT-BA, accounting for 66.5%TRR (0.117 mg/kg eq) in DAT 40 plants treated with a post emergence application of isoxaflutole. It was also the major metabolite in DAT 81 and 95 sugarcane samples (pre-emergence application): 86–94%TRR, or 0.102 and 0.138 mg/kg eq at 81 DAT or 95 DAT harvested plants, respectively. IFT-DKN was only detected in the 40 DAT harvested sugar cane plants that had received a post emergence application (2.2%TRR or 0.004 mg/kg eq). Parent IFT was not found in any of the samples. Unknown compounds (9.8–10.8%TRR) were detected in the DAT 40/81 samples (pre- and post-emergence application), but were not detected in the DAT 95 or DAT 365 samples.

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Storage stability was not addressed as the samples were analysed within 6 months after extraction and the plant parts were stored frozen (≤ –20 °C) prior to extraction and refrigerated (4 °C) between handling steps). It is noted that it took 6 months between harvest and extraction and an additional month for HPLC extraction. Isoxaflutole degrades after 3 months to IFT-DKN (see section on storage stability). This could pose a problem. As the main metabolite in this case is IFT-BA, which is stable, it is not expected that the longer storage of the samples will have influenced the results of this study significantly.

Table 16 Characterisation of residues in sugar cane following a single pre- or post-emergence application of isoxaflutole

DAT TRR (mg/kg eq)

IFT IFT-BA IFT-DKN Unknowns (polar)

Total extracted

Solids

%TRR mg/kg eq

%TRR mg/kg eq

%TRR mg/kg eq

%TRR mg/kg eq

%TRR (mg/kg eq)

%TRR (mg/kg eq)

Pre-emergence application (field rate 0.210 kg ai/ha) 81 0.1188 – – 85.93 0.102 – – 9.81 0.012 95.7

(0.1137) 4.3 (0.0051)

95 0.1473 – – 93.50 0.138 – – – – 93.5 (0.1377)

6.5 (0.096)

365 0.0008 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. Post emergence application (field rate 0.133 kg ai/ha) 40 0.1757 10.76 0.019 66.51 0.117 2.20 0.004 10.9 0.019 90.4

(0.1587) 9.6 (0.0170)

95 0.0065 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 365 0.0004 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

n.a. = not applicable; – = not detected;

Study 7

A study was conducted to demonstrate the effects of the safener cyprosulfamide (AE 0001789) on the metabolism of isoxaflutole in maize [Schulte, 2002, M-210791-01-1]. Three day old seedlings of Zea mays (maize variety: Lorenzo) plants were transferred with their primary roots into 3.5 ml vials. The roots were exposed to a nutrient solution containing 0.5 μg ai/mL as [U-14C-phenyl]-isoxaflutole alone or in combination with safener cyprosulfamide (1 μg/ml). Pure radio-labelled IFT was used without formulation. The safener cyprosulfamide was applied as WP20 formulation. The seedlings were incubated for 24 hr. During that time roots and shoots approximately duplicated their weight and an uptake of 1 ml to 2 ml of the application solution was observed. After 24 hr incubation the seedlings were transferred into beakers filled with nutrient solution (without IFT or safener). In these beakers plants grew for further 3 days in the plant chamber.

Plants treated with isoxaflutole alone had partially bleached leaves at DAT = 3, whereas the maize plants previously incubated with isoxaflutole in combination with the safener showed a clear reduction of leaf damage.

Plants were harvested at 3 DAT and material from 50 plants (shoots, seeds and roots) was used for preparation of one sample. The samples were homogenized and filtered subsequently with ACN/water (80/20, v/v), ACN/water (50/50), mixed with trifluoroacetic acid (TFA), and partitioned three times in EtOAc (shoot and root extracts only). The filtrates or acetyl extracts were subjected to radio-TLC. Metabolites were separated by their Rf values and their radioactivity was measured. The extracts were analysed to determine the levels of the two known IFT metabolites IFT-DKN and IFT-BA by combustion and LSC.

Distribution of radioactivity is shown in Table 17. After three days radioactive distribution over the plant is different for the isoxaflutole and isoxaflutole/safener combination. Planted seeds incorporated more radioactivity than shoots and roots.

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Parent IFT was not found due to the rapid conversion to the IFT-DKN metabolite. Additionally plants treated with the isoxaflutole/safener combination show a lower ratio of the IFT-DKN/IFT-BA metabolites in the shoots compared to those plants treated with isoxaflutole alone (factor 2.3, see Figure 3). In seeds and roots no differences in the ratios between the IFT metabolites could be demonstrated and only small enzymatic degradation of IFT-DKN to the inactive IFT-BA metabolite was observed.

To check whether mono-oxygenase inhibitors 1-aminobenzotriazole (ABT) and piperonyl butoxide (PBO) antagonise safener activity of cyprosulfamide also treatment solutions containing ABT or PBO were tested. After treatment the seedlings of those inhibitor-treated plants were transferred to nutrient solution which also contained ABT or PBO. The addition of mono-oxygenase inhibitors did not reduce the safener effects, indicating that mono-oxygenases are not involved in the safener effects of cyprosulfamide.

The reduced uptake of radioactivity in the shoots of plants treated with the safener combination suggests that at least one of the protective effects of the safener might be due to a reduced translocation of IFT-DKN from the root system into the shoot.

Note: Compared to the roots relatively much radioactivity is observed in the planted seeds. According to the manufacturer the radioactivity is mainly located in the transition area between root and shoot and only a small distribution in the endosperm. The accumulation in the transition area between roots and shoots could be caused by the changes in the arrangement of the vascular bundles between roots and shoots [Bayer, 2013].

Table 17 Distribution of the radioactivity (sum of IFT, IFT-DKN and IFT-BA) between the different plant parts

Isoxaflutole only treatment (% TAR)

Isoxaflutole + Safener treatment (% TAR)

Shoots 30.2 25.5 Seeds 42.1 48.3 Roots 27.8 26.2

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Figure 3 Ratio of IFT-DKN/IFT-BA for isoxaflutole and isoxaflutole/safener treatment

Overview metabolic pathway of isoxaflutole in plants

The same metabolic pathway of isoxaflutole was observed in the various metabolism studies on primary crops and rotational crops. The first hydrolytic step is the opening of the isoxazole ring to form IFT-DKN. Further hydrolytical cleavage of the carbonyl bridge and loss of the complete isoxazole moiety leads to the corresponding benzoic acid derivative (IFT-BA). The corresponding aminolysis to IFT amide could only be observed in the glyphosate/HPPD-tolerant soya beans. Application of a safener cyprosulfamide affects the breakdown of IFT-DKN to IFT-BA. The pathway is shown schematically in Figure 4.

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Figure 4 Proposed metabolic pathway of isoxaflutole in plant commodities

Environmental fate in soil

The Meeting received information on aerobic degradation in soil, photolysis on soil, and confined and field rotational crop studies. The fate and behaviour of isoxaflutole in the environment was investigated using [U-14C-phenyl]-isoxaflutole (see Figure 1).

Aerobic degradation in soil - laboratory studies

Study 1

The rate of aerobic degradation of isoxaflutole in a sandy loam soil at a temperature of 20 °C was investigated [Burr, 1996, M-240821-01-1]. Soil characteristics are reported in Table 18. Soils were stored aerobically at the test facility at 4 °C. The surface of a soil was then treated with [U-14C-phenyl]-isoxaflutole at a nominal rate of 0.2 mg ai/kg dry soil, corresponding with a field application rate of 0.20 kg ai/ha. Soil samples were incubated under aerobic conditions in the dark at 20 ± 2 °C and soil moisture of 45% of the maximum water holding capacity for up to 365 days.

Samples were taken at 0, 3 and 6 hours and at DAT = 1, 2, 3, 7, 14, 28, 59, 92, 120, 181, 273 and 365. All samples generated during the study were analysed as soon as possible, within a few days (maximum of 10 days) after generation.

The soil samples were extracted at ambient temperature using ACN/water followed by extraction with acidified (pH 3) ACN/water. Starting by day 59, samples were extracted with EtOAc in addition. Extracts were concentrated under reduced pressure by rotary evaporation at approximately

CF3

SO2CH3O

NO

CF3

OO SO2CH3

CN

CF3

O

NH2

SO2CH3

CF3

O SO2CH3

OH

Isoxaflutole (RPA201772)

IFT-DKN (RPA 202248)

IFT-amide IFT-BA (RPA 203328)

hydrolysis

aminolysis(only in soybean plant)in seed <10%TRR, and <0.01 mg/kg

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30 °C followed by reversed phase HPLC analysis and [14C]-flow-through detection as primary and TLC as confirmatory analytical method.

Table 18 Soil characteristics

Soil texture (USDA) a Sandy loam 95/04 Origin Aldhams Farm, Manningtree Essex, UK Sand (0.05–2 mm) 67.2 Silt (0.002–0.05 mm) 24.03 Clay (< 0.002 mm) 8.76 Organic Carbon (%) 2.2 Organic Matter b (%) 3.7 CEC (meq/100 g) 5.7 pH (water) 5.3 pH (CaCl2) 4.7 Moisture holding capacity @ 0.33 bar (%) 18.69 Moisture holding capacity @ 0.1 bar (%) 23.19 Maximum water holding capacity (MWHC, %) 60.28 Moisture content at incubation (%) 27.13 Bulk density (g/mL) 1.08 Microbial biomass (μg microbial carbon/g soil): —Initial 326 —After 4 months 215 —After 1 year 165

a Classification according to United States Department of Agriculture b Organic matter calculated from organic carbon content × 1.7

Isoxaflutole was degraded rapidly to form IFT-DKN and IFT-BA as major metabolites. Degradation was accompanied by the formation of minor metabolites including solids (9.13% TAR at study end, day 365) and volatile components and 14C-CO2 (1.76% TAR after 365 days of incubation). The distribution of radioactive residues is summarized in Table 19. Mass balances ranged between 94.3 and 103.8%.

Table 19 Distribution of radioactivity (% TAR) after aerobic incubation at 20 °C of soil treated with 0.2 mg ai/kg dry soil

Time Isoxaflutole IFT-DKN IFT-BA Total extracts a

Solids 14C-CO2 b Total

0 hours 92.74 1.42 nd 96.53 1.33 nd 97.8 3 hours 93.77 4.33 nd 100.35 1.62 0.01 102.0 6 hours 87.44 4.87 nd 94.47 1.66 0.01 96.1 1 day 74.55 20.01 0.38 97.24 3.23 0.01 100.5 2 days 56.02 37.82 1.42 98.62 4.19 0.01 102.8 3 days 37.74 51.97 3.52 96.26 5.61 0.02 101.9 7 days 13.49 62.13 16.54 92.70 1.50 0.07 94.3 14 days 6.73 46.19 43.30 96.71 5.19 0.23 102.1 28 days 0.37 20.02 66.98 92.31 2.85 0.61 95.8 59 days nd 9.13 81.49 93.96 6.21 0.84 101.0 92 days nd 11.18 75.31 91.02 6.15 0.95 98.1 120 days nd 7.87 83.08 96.62 5.68 1.15 103.5 181 days nd 5.90 77.16 88.23 8.15 1.23 97.6 273 days nd 5.78 82.27 94.24 8.24 1.35 103.8 365 days nd 4.44 77.27 90.95 9.13 1.76 101.8

a Other minor components accounted for < 4.2% TAR each at any sampling interval b Values include other volatile radioactivity nd = not detected

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Best fit DT50 and DT90 values of isoxaflutole and its metabolite IFT-DKN were calculated using multi-compartment kinetic models while other approaches (i.e. linear regression analysis, Timme-Frehse) resulted in poor fits to experimental data. The data are summarized in Table 20.

Table 20 Best fit values of half-lives and DT90 of isoxaflutole and its metabolites in sandy loam soil 95/04

Compound Multi compartment kinetic model DT50 (days) DT90 (days) Fit a

Isoxaflutole 2.5 8.9 –0.999 IFT-DKN 19.5 218.6 –0.997 IFT-BA –b –b –b

a A value of ± 1.000 would represent a perfect fit to the experimental data b No calculation possible due to low extent of degradation observed under conditions of the test

Study 2

The rate of aerobic degradation of isoxaflutole in two soils at a temperature of 20 °C was investigated [Ferreira et al., 1994, M-158435-01-1]. Soil characteristics are reported in Table 21. Soils were stored aerobically at the test facility at 4 °C. The surface of a soil was then treated with [U-14C-phenyl]-isoxaflutole at a nominal rate of 0.2 mg ai/kg dry soil, corresponding with a field application rate of 0.20 kg ai/ha. Soil samples were incubated under aerobic conditions in the dark at 21 ± 1 °C and soil moisture of 75% of the moisture holding capacity at 0.33 bar for up to 12 months. Samples were taken at 0, 2 and 6 hours and after 1, 2, 3, 7, 14 days and 1, 2, 3, 4, 6, 9 and 12 months of incubation after treatment (DAT). All samples generated during the study were analysed as soon as possible, within a few days (maximum of 10 days) after generation.

The soil samples were extracted at ambient temperature using ACN/water followed by extraction with acidified (pH 3) ACN/water. Starting by day 3 (clay soil) or after 1 month (sandy loam) samples were successively Soxhlet-extracted with EtOAc and hexane. Extracts were concentrated under reduced pressure by rotary evaporation at approximately 30 °C followed by reversed phase HPLC analysis and [14C]-flow-through detection as primary and TLC as confirmatory analytical method.

Table 21 Soil characteristics

Texture (USDA) a Sandy loam 93/7 Clay 93/6 Origin American Agricultural Services

Gourd Branch Road Lucama, NC, US

Coopers Shaw Road West Tilbury Marshes Essex, UK

Sand (0.05–2 mm) 69.95 11.58 Silt (0.05–0.002 mm) 23.79 38.78 Clay (< 0.002 mm) 6.26 49.64 Organic matter (%) 1.20 7.61 Organic carbon (%) 0.9 4.5 pH (water) 6.6 5.8

pH (1 M KCl) 5.7 4.6 CEC (mEq/100 g soil) 4.9 37.7 Bulk Density (g/mL) 1.54 0.88 Moisture Holding Capacity at 0.33 bar (%) 13.0 50.57 Fungi (organisms/g dry soil) 3.18 × 103 5.13 × 103

Bacteria (organisms/g dry soil) 2.9 × 106 2.18 × 106

Actinomycetes (organisms/g dry soil) 3.4 × 106 3.38 × 106

Biomass (μg microbial carbon/g soil): —Beginning of Study 122.94 989.79 —End of Study 174 504 Soil Nomenclature (Series, Order, Sub-Order) Norfolk, Ultisol, Typic

Paleudults Wallasea, Entisol, Typic Fluvaquent

Isoxaflutole

1201

a Classification according to United States Department of Agriculture

Mass balances ranged between 90.7% and 108.0% of TAR for sandy loam soil 93/7 and 90.8% to 102.9% TAR for clay soil 93/6. Results for the extraction, distribution and identification of radioactivity are given in Table 22 and Table 23.

In both test soils the isoxaflutole degraded rapidly to form IFT-DKN and IFT-BA as major metabolites. Degradation was accompanied by the formation of minor metabolites including solids (15.44% TAR for sandy loam, 22.28% for clay after 12 months of incubation) and 14C-CO2 (14.14% for sandy loam, 36.70% for clay loam after 12 months). Other volatile components were observed to a negligible extent. The distribution of radioactive residues is summarized in Tables 22 and 23.

Table 22 Isoxaflutole related residues in sandy loam soil 93/7 (% of TAR, mean values of two replicates)

Time IFT IFT-DKN IFT-BA Total extracts a


0 hours 90.28 13.90 < 0.01 104.18 1.29 – 105.5 2 hours 96.30 9.87 < 0.01 106.17 1.83 0.01 108.0 6 hours 88.39 15.22 < 0.01 103.61 1.28 0.20 105.1 1 day 55.55 45.63 < 0.01 101.62 4.06 0.10 105.8 3 day 18.18 70.67 5.39 94.24 3.37 0.18 97.8 7 day 7.13 79.80 6.25 93.62 7.36 0.13 101.1 14 day 6.49 66.36 12.27 95.60 6.75 1.08 103.4 1month < 0.01 18.40 61.78 87.46 12.29 3.55 103.3 2 months < 0.01 11.72 60.36 82.60 12.50 4.00 99.1 3 months < 0.01 15.24 56.81 85.44 11.33 4.57 101.3 4 months < 0.01 7.41 56.21 74.45 11.05 5.20 90.7 6 months < 0.01 9.17 64.58 83.44 15.07 6.96 105.5 9 months < 0.01 6.68 52.49 71.43 18.88 9.69 100.0 12 months < 0.01 5.85 46.50 72.38 15.44 14.14 102.0

a Other minor components each accounted for < 4.1% TAR at any sampling interval b Other volatile radioactivity accounted for < 0.2% TAR at any sampling interval

Table 23 Isoxaflutole related residues in clay soil 93/6 (% of TAR, mean values of two replicates)

Time IFT IFT-DKN IFT-BA Total extracts

a


0 hours 69.36 18.12 < 0.01 96.59 6.32 – 102.9 2 hours 50.91 27.77 < 0.01 93.29 5.35 0.01 98.7 6 hours 56.73 26.22 < 0.01 93.15 4.79 0.20 98.1 1 day 43.99 30.08 < 0.01 90.31 7.33 0.20 97.8 3 day 25.04 41.42 < 0.01 85.99 6.79 0.19 93.0 7 day 8.18 52.29 6.08 85.82 6.83 0.02 92.7 14 day < 0.01 47.32 11.61 84.24 10.33 0.25 94.8 1 month < 0.01 28.44 30.43 73.66 18.51 2.24 94.4 2 months < 0.01 18.79 31.00 66.65 20.38 9.13 96.2 3 months < 0.01 17.36 17.37 60.52 22.95 15.47 98.9 4 months < 0.01 14.77 13.37 45.70 23.37 21.70 90.8 6 months < 0.01 14.83 9.94 39.90 27.21 31.11 98.2 9 months < 0.01 11.75 15.31 43.05 24.82 32.46 100.3 12 months < 0.01 11.64 10.15 42.74 22.28 36.70 101.7

a Other minor components each accounted for < 6.2% TAR at any sampling interval b Other volatile radioactivity accounted for < 0.1% TAR at any sampling interval

Isoxaflutole

1202

Half-lives of isoxaflutole and its metabolite IFT-DKN in the two soils were calculated using linear regression analysis. The results are summarized in Table 24.

Table 24 Half-lives of isoxaflutole and its metabolites IFT-DKN and IFT-BA in soils sandy loam 93/7 and clay 93/6

Compound DT50 (days)

Sandy loam r2 Clay r2 Isoxaflutole 1.3 0.996 2.3 0.931 IFT-DKN 19.7 – a 37.2 – a IFT-BA 977.4 – a 288.6 – a

a Values not reported

Study 3

The rate of aerobic degradation of isoxaflutole in three soils at a temperature of 20 °C was investigated [Ferreira et al., 1996, M-240822-01-1]. Soil characteristics are reported in Table 25. Soils were stored aerobically at the test facility at 4 °C. The surface of a soil was then treated with [U-14C-phenyl]-isoxaflutole at a nominal rate of 0.2 mg ai/kg dry soil, corresponding with a field application rate of 0.20 kg ai/ha. Soil samples were incubated under aerobic conditions in the dark at 20 ± 2 °C and soil moisture of 45% of the maximum moisture holding capacity. Samples of loam 95/02 soil were removed for analysis after 0, 1, 3, 5, 8, 16, 33, 64, 101 and 120 days of incubation while samples of loamy sand 95/06 were removed after 0, 0.25, 0.75, 1.67, 7, 15, 30, 63, 91 and 120 days. Finally, samples of clay loam 95/05 were removed after 0, 0.125, 0.25, 0.375, 3, 7, 17, 46, 82 and 120 days.

Soil samples of loam 95/02 and loamy sand 95/06 were extracted by using ACN/0.01 M aqueous calcium chloride followed by extraction with acidified (pH 3) ACN/water at ambient temperature. In case radioactive residues in extracted soil were > 10% TAR, samples were successively Soxhlet-extracted with acidified (pH 3) ACN/water and EtOAc. Samples of loamy sand 95/06 and incubated for 30 days were additionally extracted with aqueous 0.01 M diaminoethanetetra-acetic acid (EDTA) solution at ambient temperature. Soil samples of clay loam 95/05 were extracted by using ACN/0.01 M aqueous calcium chloride at ambient temperature. In case radioactive residues remaining solids were > 10% TAR, samples were extracted with aqueous 0.01 M EDTA solution at ambient temperature followed by Soxhlet extraction with EtOAc. Extracts were concentrated under reduced pressure by rotary evaporation at approximately 30 °C followed by reversed phase HPLC analysis and [14C]-flow-through detection as primary and TLC as confirmatory analytical method. Selected soil extracts were investigated by HPLC-MS.

Table 25 Physico-chemical characteristics of the test soils

Texture (USDA) a Loam 95/02

Loamy Sand 95/06

Clay Loam 95/05

Origin Well Field Boarded Barns Farm Shelley, Ongar, Essex, UK

Flint Hill Farm Roysten, Hertfordshire, UK

Roos Hall Beccles, Suffolk, UK

Particle size distribution (%) Sand (2–0.063 mm) 35.78 87.07 18.98 Silt (0.063–0.002 mm) 44.19 5.58 45.46 Clay (< 0.002 mm) 20.02 7.35 35.55 Organic carbon 1.9 1.1 2.5 Organic matter b 3.2 1.9 4.3 CEC (mEq/100 g) 1.9 1.1 2.5 pH in Water 4.8 7.4 8.2 pH (0.01 M CaCl2) 4.3 6.7 7.8 Moisture Holding Capacity at 0.33 bar c (%) 19.9 24.13 29.25 Moisture Holding Capacity at 0.1 bar d (%) 25.52 28.28 62.48 Maximum Holding Capacity (%) 59.91 38.82 77.79

Isoxaflutole

1203

Texture (USDA) a Loam 95/02

Loamy Sand 95/06

Clay Loam 95/05

Origin Well Field Boarded Barns Farm Shelley, Ongar, Essex, UK

Flint Hill Farm Roysten, Hertfordshire, UK

Roos Hall Beccles, Suffolk, UK

Moisture Content During Incubation (%) 26.96 17.47 35.01 Microbial Biomass (μg/g soil): —Beginning of Study 225 191 558 —End of Study 172 175 613

a Classification according to United States Department of Agriculture b Calculated from organic carbon content x 1.7 c Equivalent to pF = 2.5 d Equivalent to pF = 0

The total recovery of radioactivity ranged 97.4–104.6% of TAR for soil loam 95/02, 91.6–102.5% for soil loamy sand 95/06 and 90.6–102.9% for soil clay loam 95/05 (mean values of two replicates).

In all test soils the isoxaflutole degraded rapidly to form IFT-DKN as a major metabolite. The further conversion proceeded via formation of IFT-BA as a major metabolite in soils loam 95/02 and loamy sand 95/06. In soil clay loam 95/05, IFT-BA was observed as a minor component (7.13% TAR by day 7 in maximum) along with the formation significant amounts of solids (31.22% by day 120) and 14C-CO2 (34.62%, day 120).

Degradation was accompanied by the formation of minor metabolites including RPA 205834. In soils loam 95/02 and loamy sand 95/06, formation of solids (9.02% for loam 95/02, 4.09% for loamy sand 95/06 after 120 days of incubation) and 14C-CO2 (1.26% for loam 95/02, 0.70% for loamy sand 95/06 after 120 days) was less pronounced indicating a slow transformation of isoxaflutole residues beyond IFT-BA under the conditions of the test. The distribution of radioactive residues is summarized in Table 26 to Table 28.

Table 26 Isoxaflutole related residues in loam soil 95/02 (% of TAR, mean values of two replicates)

Time [days]

IFT IFT-DKN IFT-BA Total extracts

a

Solids 14C-CO2 Total

0 94.62 3.68 nd 98.30 1.95 n.m. 100.3 1 80.81 14.07 nd 94.88 2.80 n.m. 97.7 3 64.37 27.00 nd 91.37 5.92 0.05 97.4 5 35.76 51.92 4.37 93.09 7.94 0.05 101.1 8 24.57 63.78 6.78 97.07 1.84 0.02 98.9 16 11.20 71.22 16.81 101.55 2.84 0.21 104.6 33 5.62 55.75 30.85 94.33 3.99 0.29 98.6 64 1.82 38.21 49.98 91.98 5.94 0.72 98.7 101 0.64 31.45 60.01 93.56 6.14 0.97 100.7 120 nd 26.23 64.07 90.30 9.02 1.26 100.6

a Other minor components including RPA 205834 each accounted for < 2.4% TAR at any sampling interval n.m. = not measured nd = not detected

Isoxaflutole

1204

Table 27 Isoxaflutole related residues in loamy sand soil 95/06 (% of TAR, mean values of two replicates)

Time [days]


a


0 93.93 2.39 nd 96.32 0.23 n.m. 96.6 0.25 70.19 27.22 nd 97.41 0.47 n.m. 97.9 0.75 42.35 58.74 0.71 101.80 0.70 n.m. 102.5 1.67 8.28 85.84 2.25 96.36 1.37 n.m. 97.7 7 nd 94.60 1.84 96.44 1.79 0.18 98.4 15 nd 85.86 4.80 90.66 1.39 0.33 92.4 30 nd 41.75 41.84 84.08 4.76 2.61 91.6 63 nd 10.44 78.21 89.82 6.55 3.13 99.5 91 nd 5.21 89.83 95.25 3.93 0.59 99.8 120 nd 4.15 90.02 95.02 4.09 0.70 100.0


Table 28 Isoxaflutole related residues in clay loam soil 95/05 (% of TAR, mean values of two replicates)

Time [days]


a


0 92.13 4.31 nd 96.45 4.48 n.m. 100.9 0.125 74.64 23.03 nd 97.66 4.83 n.m. 102.5 0.25 52.92 40.83 nd 93.75 6.01 n.m. 99.8 0.375 41.31 51.00 nd 92.31 6.98 0.05 99.3 3 nd 96.39 3.66 100.05 2.73 0.14 102.9 7 1.36 83.89 7.13 92.38 3.81 0.45 96.6 17 1.33 72.18 6.80 79.19 9.46 4.43 94.8 46 nd 45.53 2.78 53.68 21.37 16.05 93.0 82 nd 28.80 1.64 33.11 27.96 26.22 90.6 120 nd 20.24 0.72 23.61 31.22 34.62 92.2


Half-lives of isoxaflutole in the three soils were calculated using linear regression analysis assuming simple first order kinetics. For metabolites IFT-DKN and IFT-BA values for half-lives and DT90 in aerobic soil were calculated, where possible, by using the software Kinetic Modelling (KIM). The results are summarized in Table 29.

Table 29 Half-lives and DT90 of IFT and its metabolites IFT-DKN and IFT-BA in soils clay loam 95/02, loamy sand 95/06 and loam 95/05

Compound IFT Soil DT50 DT90 Correlation coefficient r2

Loam 95/05 4 days 13.1 days 0.977 Loamy Sand 95/06 11.4 hours 37.8 hours 0.983 Clay Loam 95/02 7.6 hours 25.1 hours 0.992 Compound IFT-DKN Soil DT50 DT90 Fitting criterion Loam 95/05 25 days – a –0.993 Loamy Sand 95/06 56 days – a –0.988 Clay Loam 95/02 41 days – a –0.998

Isoxaflutole

1205

Compound IFT Soil DT50 DT90 Correlation coefficient r2

Compound IFT-BA Loam 95/05 –c – b – b Loamy Sand 95/06 –c – b – b Clay Loam 95/02 –c – b – b

a Not calculated b Not calculable, because study period was too short for this metabolite

Soil photolysis

The rate of photolysis was investigated on a 93/22/1 sandy loam soil [Ferreira, 1994, M-158351-01-1]. The soil was surface-treated with [U-14C-phenyl]-isoxaflutole at an exaggerated rate of 36.5 μg/sample, equivalent to 0.645 kg ai/ha. Soil samples were irradiated under aerobic conditions at 25 ± 1 °C and soil moisture of 75% of the moisture holding capacity at 0.33 bar. Another set of soil samples was incubated under aerobic conditions in the dark at 25 ± 1 °C and soil moisture of 75% of the moisture holding capacity at 0.33 bar to serve as dark controls. Samples of irradiated soil and dark controls were removed for analysis after 0, 0.125, 0.25, 0.67, 1, 2, 7, 14, 21 and 31 days of incubation. The characteristics of the test soils are given in Table 30.

Soil samples were extracted with aqueous ACN (day 0 samples) followed by extraction with acidified (pH 3) ACN/water at ambient temperature (samples of day 1, 2, 7, 14, 21 and 31). Dark controls of day 31 were Soxhlet-extracted using EtOAc, followed by hexane and 1 M HCl. Extracts were concentrated at approximately 31 °C followed by reversed phase HPLC analysis and 14C-flow-through detection as primary and TLC as confirmatory analytical method. Selected soil extracts were investigated by HPLC-MS.

Table 30 Physico-chemical characteristics of test soil

Soil texture (USDA) a Sandy loam 93/22 Origin Field Research Facility, American Agricultural Services,

Lacama, NC, US

Sand (0.05–2 mm) 53.98 Silt (0.002–0.053 mm) 40.80 Clay (< 0.002 mm) 5.23 Organic Carbon (%) 1.3 Organic Matter b (%) 2.2 CEC (meq/100 g) 5.7 pH (water) 7.07 pH (0.01 M CaCl2) 5.54 Moisture holding capacity @ 0.33 bar (%) 11.00 Bulk density (g/mL) 1.59 Microbial activity (organisms per g of dry soil): —Fungi 4.53 × 103 —Bacteria 2.63 × 106 —Actinomycetes 1.365 × 106

a Classification according to United States Department of Agriculture b Organic matter calculated from organic carbon content × 1.7

The total recovery of radioactivity ranged from 88.5–101.4% of TAR for irradiated soil samples and from 91.6–101.5% for dark controls (mean values of two replicates). Isoxaflutole degraded rapidly to form IFT-DKN and IFT-BA as major metabolites in irradiated samples as well as in dark controls. Both metabolites have been observed as transformation products in aerobic soil degradation, IFT-DKN being the result of (abiotic and biotic) hydrolysis and IFT-BA to result from microbial degradation of IFT-DKN. In irradiated samples, solids were formed to a low extent (7.63% by day 31) accompanied by insignificant levels of 14C-CO2 (0.13%, day 31). For dark controls

Isoxaflutole

1206

formation of solids and mineralisation to 14C-CO2 was also low being at a similar level as observed for irradiated samples (NER: 4.02%, 14C-CO2: 0.06%, both day 31).

Since the decline of parent compound and the extent of formation of metabolites observed in this experiment was virtually the same for both, i.e. irradiated samples and dark controls, there was no observation of components formed specifically from photo-transformation processes. This allows for the conclusion that the extent of photolytic degradation was insignificant. The distribution of radioactive residues is summarized in Table 31 for irradiated samples and in Table 32 for dark controls.

Table 31 Isoxaflutole related residues in irradiated samples of loam soil 93/22 (% of TAR, mean values of two replicates)

Time [days]


Solids 14C-CO2 a Total

0 95.40 4.04 < 0.01 99.43 0.20 n.m. 99.6 0.125 77.81 19.62 < 0.01 97.42 0.83 < 0.01 98.3 0.25 70.73 28.11 < 0.01 98.84 1.17 < 0.01 100.0 0.67 53.21 41.02 < 0.01 94.22 2.11 < 0.01 96.3 1 44.03 41.51 < 0.01 87.74 0.76 < 0.01 88.5 2 31.86 53.31 1.53 90.42 1.00 < 0.01 91.4 7 4.60 78.77 4.48 93.95 2.44 0.02 96.4 14 1.50 74.65 12.14 95.98 4.04 0.03 100.1 21 < 0.01 69.79 15.45 93.49 5.15 0.08 98.7 31 b < 0.01 58.42 25.78 93.58 7.63 0.13 101.4

a Other volatile components accounted for 0.01% or less at any sampling interval b Mean values of four replicates n.m. = not measured

Table 32 Isoxaflutole related residues in dark controls of loam soil 93/22 (% of TAR, mean values of two replicates)

Time [days]


Solids 14C-CO2 a Total

0 95.40 4.04 < 0.01 99.43 0.20 n.m. 99.6 0.125 64.39 30.62 < 0.01 95.00 0.80 < 0.01 95.8 0.25 67.60 28.14 < 0.01 95.74 1.18 < 0.01 96.9 0.67 59.26 34.60 < 0.01 93.86 1.99 < 0.01 95.9 1 33.11 56.98 < 0.01 92.13 0.84 < 0.01 93.0 2 31.87 55.10 1.56 92.58 1.03 < 0.01 93.6 7 4.53 69.69 6.85 87.86 3.70 0.02 91.6 14 1.52 68.59 14.79 93.85 5.36 0.02 99.2 21 < 0.01 56.57 27.05 92.31 9.14 0.04 101.5 31 b < 0.01 40.67 36.77 93.00 4.02 0.06 97.1

a Other volatile components accounted for 0.01% or less at any sampling interval b Mean values of four replicates n.m. = not measured

Half-lives of isoxaflutole derived from irradiated soil and after incubation in the dark were calculated using linear regression analysis assuming simple first order kinetics. The results are summarized in Table 33.

Table 33 Half-lives of isoxaflutole in irradiated and dark control samples in soil sandy loam 93/22

Compound IFT DT50 DT90 r2 Irradiated 22.8 hours – a not reported Dark controls 19.7 hours – a not reported

Isoxaflutole

1207

a Not calculated

Proposed degradation pathway of isoxaflutole in/on soil

The first hydrolytic step in the aerobic degradation in soils is the opening of the isoxazole ring to form IFT-DKN. Further hydrolytical cleavage of the carbonyl bridge and loss of the complete isoxazole moiety leads to the corresponding benzoic acid derivative (IFT-BA). The proposed route of degradation is shown schematically in Figure 5. These same metabolites are also found in primary and rotational crops, as a result of uptake from the soil or metabolism within the plant.

N

O SO2CH3

OCF3

O SO2CH3

CF3

O

CN

OH

O SO2CH3

CF3

Isoxaflutole, IFTRPA 201772

Diketonitrile, DKNRPA 202248

Benzoic acid, BARPA 203328

Non-Extractable Residues + CO2 Figure 5 Proposed route of degradation of isoxaflutole in/on soil under aerobic conditions

Confined rotational crop studies

The metabolism of isoxaflutole was investigated in rotational crops that were sown in pre-treated soil [Hampton and Pettaway, 1995b, M 192308-01-1]. The treatment and cultivation of crops were conducted in an outdoor fenced facility in Lucama, NC, USA in 1993–1994. The application was done as soil surface pre-emergence or as pre-plant incorporated application, at actual application rates of 0.213 kg ai/ha as [U-14C-phenyl]-isoxaflutole. Each treatment (control, soil surface or incorporated application) was performed with an isoxaflutole solution in ACN/water (1/1, v/v) in a tank filled with a lower layer of gravel and a supernatant layer (35–45 cm) of sandy loam soil (1.8% om, pH 6.3, CEC 7.9 meq/100 g, 14.6% MWHC at 1/3 bar). Radish (var White Icicle), lettuce (var Butter crunch), grain sorghum (var Pioneer Hybrid 8222), mustard (var Southern Giant Curled) and winter wheat (var Pioneer 2548) were planted in the tanks. Radish, lettuce and grain sorghum were planted at PBI 34 days, radish, mustard and winter wheat at PBI 123 days and radish, lettuce and grain sorghum at PBI

Isoxaflutole

1208

365 days. Lettuce at the last rotational interval needed replanting at 375 days due to a seedling infectious disease which reduced the stand. Prior to each planting, the soil was tilled to depth of 5–10 cm. In each rotation, one sample was taken at immature (1 to 3 weeks before maturity) and mature stages of the plants. Mature wheat and sorghum was separated in grain and straw/fodder. Radish plants were shaken to remove adhering soil and subsequently separated in roots and tops. Sample sizes were: 0.048–0.23 kg immature lettuce, 0.19–0.68 kg immature radish leaf, 0.038–0.084 kg immature radish roots, 1.1–1.4 kg sorghum forage, 0.3–1.0 kg mature lettuce, 3.3–4.6 kg mature radish leaf, 2.2–4.0 kg mature radish root, 0.69–1.0 sorghum grain, 2.4–3.2 kg sorghum fodder. Soil sample cores of 0–15 and 15–30 cm depth were sampled at the time of application and at each planting. Soil and plant samples were stored at -3 ºC or lower for 434–741 days.

Total radioactive residues (TRR) were determined in homogenised samples of crops and soil by combustion and LSC. Crop samples with TRRs greater than 0.01 mg/kg eq were subsequently extracted with hexane/EtOAc (9:1 v/v), ACN, water (pH 5.5) and ACN/0.2 M HC1 (1:1 v/v). The pH for the water extraction was adjusted to pH 5.5, because at this pH hydrolysis of IFT or isoxaflutole related conjugates did not occur. Radioactive residues in extracts and remaining solids were quantified by (combustion) LSC. For samples with appropriate levels of radioactivity, the water and ACN/0.2 M HCl extracts were partitioned against EtOAc and both layers again analysed by LSC. Organic layers containing > 10% TRR or > 0.01 mg/kg eq were concentrated and subsequently analysed by HPLC with radioactive detection and co-chromatography of standards for IFT, IFT-DKN and IFT-BA. Parent and metabolites were confirmed by HPLC-MS-MS with APCI when feasible using standards for IFT, IFT-DKN, IFT-BA and RPA 205834.

Based on application rate, expected levels are 0.11 mg/kg eq in the top 0–15 cm soil. The TRR in the top 0–15 cm treated soil significantly decreased with time from 0.07–0.08 mg/kg eq at day 0 to a plateau of 0.02–0.03 mg/kg eq. The concentration at 15–30 cm was fairly stable at 0.01 mg/kg eq indicating very minor leaching of radioactivity. Radio-activity was not further characterized.

The results in rotational crops are shown in Table 34 (pre-plant incorporated) and Table 35 (pre-emergence). The highest total radioactive residue (TRR) of 0.24 mg/kg eq was found in sorghum forage of the first rotation after pre-plant incorporated soil application.

The metabolic profile was similar in all crops of the study. IFT was detected by HPLC-MS-MS in trace amounts (< 0.001 mg/kg eq) in radish leaves, sorghum grain and sorghum fodder of the first rotation (34 days) and wheat forage of the second rotation (123 days). IFT-BA represented the major compound and was present in the RACs of the first (34 days), second (123 days) and third rotation (365 days) at levels between < 0.001–0.11 mg/kg eq. IFT-DKN was only found in radish leaves and sorghum grain at levels up 0.005 mg/kg eq in the first rotation (34 days). RPA 205834 was detected by HPLC-MS-MS in trace amounts (< 0.001 mg/kg eq) in mature lettuce of the first rotation (34 days). A fourth metabolite (U1) was found in the RACs of the first (34 days), second (123 days) and third rotation (365 days) at levels between < 0.001–0.022 mg/kg eq. This metabolite was analysed by HPLC-MS-MS after extensive purification and a relative molecular weight of 192 was determined. The polarity of this metabolite, as evidenced by its relative retention time, suggests it is a carboxylic acid degradation product of IFT-BA with the structural formula CF3-C6H4-COOH (4-trifluoromethyl benzoic acid).

Based on storage stability study 6, it was concluded that after 23 months of storage at –20 °C, IFT partially converts to IFT-DKN and RPA 203328 in sorghum forage, sorghum fodder, sorghum grain, lettuce, radish leaves and radish roots. Total residues (sum of IFT, IFT-DKN and IFT-BA) remained stable for at least 23 months in each matrix tested. Results from the aerobic soil degradation studies showed that isoxaflutole had a short half-life (30 hrs) in sandy loam soil and that IFT-BA was the predominant residue in soil after 1 month. These findings suggest that the likelihood that isoxaflutole was present in the soil in the PBI 34 day is low and that IFT-BA is the main metabolite present for plant uptake.

Isoxaflutole

1209

Table 34 TRR and nature of residues in rotational crops after pre-plant-soil incorporated application

PBI days (rotation number)

Matrix DAP DAT TRR mg/kg eq

IFT %TRR

IFT-DKN % TRR (mg/kg eq)

IFT-BA %TRR (mg/kg eq)

U1 %TRR (mg/kg eq)

Solids %TRR

Total %TRR

34 (1st)

Immature radish leaf

21 55 0.014 0 < 0.001)

7.1 (0.001)

78.6 (0.011)

0 (< 0.001)

9.2 94.9

Mature radish leaf

41 75 0.019 0 (< 0.001)

26.3 (0.005)

36.8 (0.007)

0 (< 0.001)

5.5 68.6

Immature radish root

21 55 0.008 NE

– – – – – –

Mature radish root

41 75 0.003 NE

– – – – – –

34 (1st)

Immature lettuce leaf

41 75 0.042 0 (< 0.001)

0 (< 0.001)

74.4 (0.029)

23.8 (0.010)

3.3 101.5

Mature lettuce leaf

49 83 0.022 0 (< 0.001)

0 (< 0.001)

59.1 (0.013)

31.8 (0.007)

6.3 97.2

34 (1st)

Sorghum forage

41 75 0.24 0 (< 0.001)

0 (< 0.001)

100 (0.24)

0 (< 0.001)

1.9 101.9

Mature sorghum fodder

134 168 0.13 0 (< 0.001)

0 (< 0.001)

74.6 (0.097)

0 (< 0.001)

7.1 81.7

Mature sorghum grain

134 168 0.12 0 (< 0.001)

0.8 (0.001)

67.2 (0.080)

10.1 (0.012)

11.4 89.5

123 (2nd)


45 168 0.002 NE

– – – – – –

Mature radish leaf

58 181 0.002 NE

– – – – – –


45 168 0.001 NE

– – – – – –

Mature radish root

58 181 0.001 NE

– – – – – –

123 (2nd)

Immature mustard leaf

45 168 0.004 NE

– – – – – –

Mature mustard leaf

58 181 0.002 NE

– – – – – –

123 (2nd)

Immature wheat forage

133 256 0.013 0 (< 0.001)

0 (< 0.001)

100 (0.013)

0 (< 0.001)

5.3 105.3

Mature wheat straw

239 362 0.030 0 (< 0.001)

0 (< 0.001)

70.0 (0.021)

0 (< 0.001)

25.7 95.7

Mature wheat grain

239 362 0.018 a 0 (< 0.001)

0 (< 0.001)

100 a

(0.018) 0 (< 0.001)

6.4 a

(0.0012) 106.4 a

365 (3rd)


28 393 0.008 NE

– – – – – –

Mature radish leaf

38 403 0.011 a 0 (< 0.001)

0 (< 0.001)

18.2 a

(0.002) 81.8 a

(0.009) 3.9 (< 0.001)

103.9 a


28 393 0.002 NE

– – – – – –

Mature radish root

38 403 0.001 NE

– – – – – –

375 (3rd)


51 426 0.006 NE

– – – – – –

Mature lettuce leaf

71 446 0.005 NE

– – – – – –

365 (3rd)

Sorghum forage

38 403 0.051 0 (< 0.001)

0 (< 0.001)

31.4 (0.016)

33.3 (0.017)

3.4 68.1

Mature sorghum

110 475 0.041 a 0 (< 0.001)

0 (< 0.001)

46.3 a

(0.019) 46.3 a

(0.019) 7.3 a

(0.003) 99.9 a

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IFT %TRR

IFT-DKN % TRR (mg/kg eq)


U1 %TRR (mg/kg eq)

Solids %TRR

Total %TRR

fodder Mature sorghum grain

110 475 0.007 NE

– – – – – –

NE = not extracted due to matrix TRR being at or below 0.01 mg/kg eq a Adapted by the reviewer, based on individual values for metabolites. Original TRR values measured by combustion were 0.017 mg/kg eq for mature wheat grain PBI 123 days, 0.010 mg/kg eq for mature radish leaves PBI 365 days and 0.029 mg/kg eq for mature sorghum fodder PBI 365 days.

Table 35 TRR and nature of residues in rotational crops after pre-emergence soil surface application



RPA 201722 %TRR (mg/kg eq)

IFT-DKN %TRR (mg/kg eq)


U1 %TRR (mg/kg eq)

Solids %TRR

Total %TRR

34 (1st)


21 55 0.015 0 (< 0.001)

0 (< 0.001)

86.7 (0.013)

0 (< 0.001)

12.4 99.1

Mature radish leaf

41 75 0.011 0 (< 0.001)

27.3 (0.003)

81.8 (0.009)

0 (< 0.001)

5.4 114.5


21 55 0.010 NE

– – – – – –

Mature radish root

41 75 0.003 NE

– – – – – –

34 (1st)


41 75 0.056 a 0 (< 0.001)

0 (< 0.001)

94.6 a

(0.053) 0 (< 0.001)

5.9 a

(0.003) 100.5

Mature lettuce leaf

49 83 0.030 0 (< 0.001)

0 (< 0.001)

63.3 (0.019)

26.7 (0.008)

5.3 90.0

34 (1st)

Sorghum forage 41 75 0.126 0 (< 0.001)

0 (< 0.001)

86.5 (0.11)

0 (< 0.001)

2.3 88.8


134 168 0.037 0 (< 0.001)

0 (< 0.001)

24.3 (0.009)

47.1 (0.016)

7.1 78.5


134 168 0.038 0 (< 0.001)

5.3 (0.002)

63.2 (0.024)

15.8 (0.006)

12.6 91.6

123 (2nd)


45 168 0.002 NE

– – – – – –

Mature radish leaf

58 181 0.002 NE

– – – – – –


45 168 0.001 NE

– – – – – –

Mature radish root

58 181 < 0.001 NE

– – – – – –

123 (2nd)

Immature mustard leaf

45 168 0.004 NE

– – – – – –

Mature mustard leaf

58 181 0.002 NE

– – – – – –

123 (2nd)

Wheat forage 133 256 0.009 0 (< 0.001)

0 (< 0.001)

55.6 (0.005)

33.3 (0.003)

4.6 93.5

Mature wheat straw

239 362 0.042 0 (< 0.001)

0 (< 0.001)

73.8 (0.031)

0 (< 0.001)

21.9 95.7

Mature wheat grain

239 362 0.015 0 (< 0.001)

0 (< 0.001)

100 (0.015)

0 (< 0.001)

7.9 107.9

365 (3rd)


28 393 0.010 NE – – – – – –

Mature radish leaf

38 403 0.010 0 (< 0.001)

0 (< 0.001)

0 (< 0.001)

100 (0.010)

4.8 104.8


28 393 0.002 NE – – – – – –

Mature radish root

38 403 0.001 NE – – – – – –

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RPA 201722 %TRR (mg/kg eq)

IFT-DKN %TRR (mg/kg eq)


U1 %TRR (mg/kg eq)

Solids %TRR

Total %TRR

375 (3rd)


51 426 0.008 NE – – – – – –

Mature lettuce leaf

71 446 0.010 NE – – – – – –

365 (3rd)

Sorghum forage 38 403 0.030 0 (< 0.001)

0 (< 0.001)

13.3 (0.004)

46.7 (0.014)

2.8 62.8


110 475 0.029 0 (< 0.001

0 (< 0.001)

6.9 (0.002)

75.9 (0.022)

3.6 86.4


110 475 0.004 NE

– – – – – –

NE = not extracted due to matrix TRR being at or below 0.01 mg/kg eq a Adapted by the reviewer, based on individual values for metabolites. Original TRR values measured by combustion were 0.037 mg/kg eq for mature lettuce leaves PBI 23 days, 0.010 mg/kg eq for mature radish leaves PBI 365 days, and 0.029 mg/kg eq for mature sorghum fodder PBI 365 days.

Field rotational crop studies

A study was conducted in the USA during the 1997–1999 growing seasons to determine the isoxaflutole related residues in rotational crops [Mickelson, 2000, M-240710-01-1]. One trial was established in Illinois and one in Ohio. In each trial isoxaflutole was applied once at a rate of 0.154–0.161 kg ai/ha in a minimum spray volume of 93.5 L/ha to bare ground as pre-plant or pre-emergence application of the primary crop (maize). Maize plants were harvested and/or removed from the plots prior to the planting of the rotational crops [Bayer, 2013]. The study was designed to investigate three representative rotational crop groups (leafy vegetables (mustard and soya bean), root crops (sugar beet, turnip, and radish) and small grains (winter wheat and sorghum)) at four plant-back intervals (30 days, 100–120 days, 150–180 days and 365 days). Each test site selected the variety best suited for the growing area within each crop group. Replicate field samples of the following commodities were sampled for residue analysis: mustard (leaves), sugar beet (top and root), turnip (top and root), radish (top and root), wheat (forage, hay, straw and grain), soya bean (forage, hay, wheat and grain) and sorghum (forage, hay, wheat and grain). Sample sizes were at least 0.5 kg for hay, straw and fodder and at least 1 kg for grains, seeds and cereal forage (except 0.45–0.68 kg for wheat grain). Sample sizes were 1.1–3.0 kg for mustard leaves, 1.4–3.6 kg for turnip roots + tops, 0.45–2.5 kg for radish roots + tops, 2.5–18 kg for sugar beets + tops, but the number of items was not indicated. All samples were stored at –5 ºC or lower for a maximum period of 721 days (24 months).

IFT, IFT-DKN and IFT-BA were determined according to modification A and B of HPLC-MS-MS method CAL#019-03. Average concurrent recovery data for modification A (n=1/level, at 0.01–0.05–0.5 mg/kg) were within 70–120% for all analytes in wheat hay, wheat grains, wheat straw, soya bean seeds, soya bean hay, sorghum grains, sorghum fodder. Recoveries are not satisfactory for IFT-BA in wheat hay (62%, n=1, 0.01 mg/kg), wheat straw (68%, n=1, 0.01 mg/kg) and soya bean seeds (64%, n=1, 0.01 mg/kg). No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control samples. Average concurrent recovery data for modification B were within (at n=1/level, 0.01–0.05-0.5 mg/kg) were within 70–120% for all analytes in mustard leaves, turnip roots, turnip tops, wheat forage, soya bean forage, radish roots, radish tops and sorghum forage. Recoveries are not satisfactory for IFT-BA in turnip tops (69%, n=1, 0.01 mg/kg) and wheat forage (69%, n=1, 0.01 mg/kg). No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control samples.

The average sample results are summarized in Table 36. None of the analysed samples showed IFT or its metabolite IFT-DKN greater than the LOQ of 0.01 mg/kg. The same applies to metabolite IFT-BA, except that it was found in samples of soya bean hay (0.010 mg/kg) and soya bean forage (0.038 mg/kg) in one trial in Ohio.

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Table 36 Results of field rotational crop trials conducted with isoxaflutole in the USA

Location Year

Rotational Crop Variety

PBI (days)

Portion analysed

GS at sampling

DAT (days)

Residues (mg/kg)

IFT IFT-DKN IFT-BA

Pre-emergence application for primary crop: Maize (planted 2 June 1997) Formulation: 75 WG, 1 application (3 June 1997); rate: 0.159 kg ai/ha and 0.170 kg ai/hL Soil: silt loam, 2.6% om, pH 6.7, CEC 7.8 meq/100 g; 31% moisture at 1/3 bar Clinton IL, USA 1997/1998

Soya bean V397RR

29 forage 6–7 node

79 n.a. n.a. n.a.

hay Mid bloom 94 < 0.01 < 0.01 < 0.01

seed Maturity 135 n.a. n.a. n.a.

Sugar beets variety not stated

tops Maturity 148 n.a. n.a. n.a. roots Maturity 148 n.a. n.a. n.a.

Sorghum Dekalb DK28E

forage Soft dough 111 n.a. n.a. n.a. grain Maturity 148 n.a. n.a. n.a. stover Maturity 148 n.a. n.a. n.a.

Mustard Florida Broadleaf

104 leaves 10–12 leaf 167 < 0.01 < 0.01 < 0.01

Turnips Purple Top White Globe

tops Early bulb 167 < 0.01 < 0.01 < 0.01 roots Early bulb 167 < 0.01 < 0.01 < 0.01

Winter Wheat Pioneer 2571

120 forage BBCH 36 324 < 0.01 < 0.01 < 0.01 hay Full bloom 344 < 0.01 < 0.01 < 0.01 grain Maturity 381 < 0.01 < 0.01 < 0.01 straw Maturity 381 < 0.01 < 0.01 < 0.01

Soya bean Pioneer 9363

365 forage 6–7 node 413 < 0.01 < 0.01 < 0.01 hay R3–R4 449 < 0.01 < 0.01 < 0.01 Seed/ grain

Maturity 493 < 0.01 < 0.01 < 0.01

Radish Cherry Bell

tops Normal harvest

426 < 0.01 < 0.01 < 0.01

roots Normal harvest

426 < 0.01 < 0.01 < 0.01

Sorghum Northrup King K5310

forage Soft dough 456 < 0.01 < 0.01 < 0.01 grain Maturity 493 < 0.01 < 0.01 < 0.01 stover Maturity 493 < 0.01 < 0.01 < 0.01

Pre-plant application for primary crop: Maize (planted 13 April and 12 May 1998) Formulation: 75 WG 1 application (2 April 1998); use rate: 0.161 kg ai/ha; 0.172 kg ai/hL Soil: silt loam, 2.6% om, pH 6.7, CEC 7.8 meq/100 g; 31% moisture at 1/3 bar Clinton IL, USA 1997/1998


151 leaves Vegetative 190 < 0.01 < 0.01 < 0.01

Radish Cherry Bell

tops Maturity 190 < 0.01 < 0.01 < 0.01 roots Maturity 190 < 0.01 < 0.01 < 0.01

Winter Wheat Clark

180 forage Starting to join

376 < 0.01 < 0.01 < 0.01

hay Dough 414 < 0.01 < 0.01 < 0.01 grain Normal

harvest 442 < 0.01 < 0.01 < 0.01

straw Normal harvest

442 < 0.01 < 0.01 < 0.01

Pre-plant application for primary crop: Maize (planted 22 May 1997) Formulation: 75 WG 1 application (22 May 1997) Use rate: 0.158 kg ai/ha; 0.169 kg ai/hL Soil: clay loam, 2.2% om, pH 7.2, CEC 10.9 meq/100 g; 26% moisture at 1/3 bar Fayette OH, USA 1997/1998

Soya bean Asgrow 3701

30 forage V3 60 < 0.01 < 0.01 0.010

hay R3 begin. pod

88 < 0.01 < 0.01 0.038

grain Maturity 144 n.a. n.a. n.a. Sugar Beets tops Maturity 144 n.a. n.a. n.a.

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Location Year

Rotational Crop Variety

PBI (days)

Portion analysed

GS at sampling

DAT (days)

Residues (mg/kg)

IFT IFT-DKN IFT-BA

variety not stated roots Maturity 144 n.a. n.a. n.a. Sorghum RS200E

forage Soft/hard dough

116 n.a. n.a. n.a.

grain Maturity 181 n.a. n.a. n.a. stover Maturity 181 n.a. n.a. n.a.


119 leaves Maturity 180 < 0.01 < 0.01 < 0.01

Turnips Purple Top White Globe

tops Root development

180 < 0.01 < 0.01 < 0.01 roots 180 < 0.01 < 0.01 < 0.01

Winter Wheat forage 6–8 in./& flag leaf

342 < 0.01 < 0.01 < 0.01

hay Dough 392 < 0.01 < 0.01 < 0.01 grain Maturity 413 < 0.01 < 0.01 < 0.01 straw Maturity 413 < 0.01 < 0.01 < 0.01

Soya bean CX368

365 forage R2–full bloom

421 < 0.01 < 0.01 < 0.01

hay Maturity 436 < 0.01 < 0.01 < 0.01 Seed/ grain

Maturity 498 < 0.01 < 0.01 < 0.01

Radish Crimson Globe

tops Maturity 419 < 0.01 < 0.01 < 0.01 roots Maturity 419 < 0.01 < 0.01 < 0.01

Sorghum RS200E

forage Dough 466 < 0.01 < 0.01 < 0.01

grain Maturity 515 < 0.01 < 0.01 < 0.01 stover Maturity 515 < 0.01 < 0.01 < 0.01

Pre-plant application for primary crop: maize (planted 14 May 1998) Formulation: 75 WG, 1 application (2 April 1998) Use rate: 0.154 kg ai/ha; 0.165 kg ai/hL Soil: clay loam, 2.2% om, pH 7.2, CEC 10.9 meq/100 g; 26% moisture at 1/3 bar Fayette OH, USA 1997/1998


166 leaves Maturity 231 < 0.01 < 0.01 < 0.01

Radish China Rose

180 tops Maturity 244 < 0.01 < 0.01 < 0.01 roots Maturity 244 < 0.01 < 0.01 < 0.01

Winter Wheat forage Feeks 6 385 < 0.01 < 0.01 < 0.01 hay Feeks 11.1–

11.2 432 < 0.01 < 0.01 < 0.01

grain Maturity 449 < 0.01 < 0.01 < 0.01 straw Maturity 449 < 0.01 < 0.01 < 0.01

GS = growth stage; n.a: not analysed.

Environmental fate in water/sediment systems

No data submitted. Not relevant for the present intended use.

RESIDUE ANALYSIS

The Meeting received information on enforcement/monitoring methods for the determination of isoxaflutole and its metabolites in plant and animal commodities. In addition the Meeting received information on analytical methods for the determination of isoxaflutole and its metabolites as used in the various study reports (supervised residue trials, storage stability studies, processing studies, feeding studies). The analytical residue methods have been evaluated according to the guidance provided by OECD (Series on Pesticides number 39) as indicated on page 25 of the FAO manual 2009.

Isoxaflutole

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Validation results are required for every commodity submitted for MRL-setting: at least one full validation for a commodity within the five defined crop groups (high acid content, high water content, high oil content, high protein content, high starch content) and a reduced validation for every other commodity within a certain crop group. Where validation results do not meet the criteria given below, this is indicated.

When the analytical method is validated according to a full validation scheme, it means that:

� at least five recovery experiments per level were conducted on at least two levels (LOQ and 10× LOQ) and average recovery per level was shown to be between 70–120% and the relative standard deviation (RSDr or CV) per level was shown to be < 20%

� at least two control samples were analysed and were shown to be below 0.3× LOQ � the calibration was conducted with at least five single points or at least three duplicate points

and was shown to be linear (either standards in solvent or matrix matched standards).

When the analytical method is validated according to a reduced validation scheme, it means that:

� a full validation is available for a crop in the same crop group (high acid content, high water content, high oil content, high protein content, high starch content)

� at least three recovery experiments per level were conducted on at least two levels (LOQ and 10× LOQ) and the average recovery per level was shown to be between 70–120% and the relative standard deviation (RSDr or CV) per level was < 20%

� at least two control samples were analysed and shown to be below 0.3× LOQ � the calibration was conducted with at least five single points or at least three duplicate points

and was shown to be linear (only relevant for matrix matched standards; standards in solvent are already covered by full validation).

Analytical methods for enforcement in plant commodities

HPLC-MS-MS method 01021

HPLC-MS-MS method 01021 is intended as enforcement method for the determination of parent isoxaflutole and its diketonitrile metabolite IFT-DKN in/on plant material [Schoening, 2007, M-282394-02-1]. Plant material was extracted with MeOH/1% formic acid solution (9/1, v/v) using a blender. After filtration of the extract the solution was made up to volume, diluted and subjected to HPLC-MS-MS. Isoxaflutole and IFT-DKN were quantified against external matrix-matched mixed standards. Two MRM transitions were monitored for each analyte and each matrix tested (m/z 360 → 251 for quantification and m/z 360 → 220 for confirmation of IFT and m/z 358 → 79 for quantification and m/z 358 → 64 for confirmation of IFT-DKN). Analyte levels were expressed as IFT equivalents.

HPLC-MS-MS Method 01021 was validated according to the full validation scheme for commodities with high acid content (orange fruit), high water content (tomato fruit), high starch content (wheat grains) and high oil content (rape seeds) [Schoening, 2007, M-282394-02-1]. The validation was performed for the quantification transition ions as well as for the confirmation transition ions. Mixed analyte solutions were used for fortification of samples. Average recoveries at 0.01 mg/kg and 0.1 mg/kg were within 70–120% limits and RSDs were within 20%. Linearity was observed in a range from 0.125–5.00 μg/L on all mass transitions (equivalent concentration in the samples not stated). The correlation coefficients for a 1/× weighted regression were r> 0.99 using five triplicate matrix matched standards. No interfering peaks > 0.3LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts. The valid LOQ was 0.01 mg/kg for each commodity.

HPLC-MS-MS Method 01021 was validated according to the full validation scheme by an independent laboratory (ILV) for tomato fruit and wheat grain [Class, 2007, M-286476-01-1]. Slight modifications were introduced: 0.1% formic acid was used instead of 1% formic acid. The validation was performed for the quantification transition ions as well as for the confirmation transition ions.

Isoxaflutole

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Mixed analyte solutions were used for fortification of samples. Average recoveries at 0.01 mg/kg and 0.1 mg/kg were within 70–120% limits and RSDs were within 20%. Linearity (coefficient of correlation r> 0.99) was observed in the range of 0.050–5.0 ng/mL for each analyte using six single matrix matched standards. The equivalent concentration in the samples was not stated. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control samples. The valid LOQ was 0.01 mg/kg for each commodity.

Conclusion: The method is considered valid for use as enforcement method in plant material with an LOQ of 0.01 mg/kg for isoxaflutole and IFT-DKN.

Analytical methods for enforcement in animal commodities

QuEChERS multi-residue method

The official multi-residue method QuEChERS was validated for the individual determination of isoxaflutole and its metabolite IFT-DKN in animal commodities. No multiresidue methods were available for determination of isoxaflutole related residues in plant commodities.

In the QuEChERS method (also named BCS method 01300/M009) animal commodities were extracted with acetonitrile and water (1:1, v/v) [Winter and Amann, 2013, M454397-01-1]. Samples with low water content (< 80%) require the addition of water before the initial extraction to get a total of approximately 10 g of water. After addition of magnesium sulphate, sodium chloride and buffering citrate salts, the mixture was shaken intensively and centrifuged for phase separation. An aliquot of the extract was taken, diluted and subjected to HPLC-MS-MS. Two MRM transitions were monitored for isoxaflutole: 358.0 to 277.9 for quantification and 358.0–78.9 for confirmation. Two MRM transitions were monitored for IFT-DKN: 358.0 to 277.9 for quantification and 358.0–78.9 for confirmation.

The validation was conducted on milk, eggs, bovine meat, bovine fat, bovine liver and bovine kidney [Winter and Amann, 2013, M454397-01-1]. To avoid the degradation of the isoxaflutole parent compound, hydrochloric acid was added to the bovine meat matrix before spiking and extraction of the samples. Matrix effects of ≥ 20% were measured for isoxaflutole and IFT-DKN in kidney and for isoxaflutole in liver. For milk, eggs, meat, fat and liver (IFT-DKN only) minor matrix effects (< 20%) were detected. Therefore, for all tested matrices, quantification was performed with matrix-matched standards. Average recoveries at 0.01 and 0.10 mg/kg (five replicates per level) were within 70–120% limits and RSDs were within 20% for each analyte and for both MRM transitions. Linearity was observed in the range of 0.25–60 ng/mL for IFT and IFT-DKN matrix matched standards. The equivalent concentration in the samples was not stated. The coefficient of determination was R2> 0.99 using six single matrix matched standards. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts. The valid LOQ was 0.01 mg/kg in milk, eggs, meat, fat, liver and kidney. Two MRM transitions were validated for all matrices and therefore an additional confirmatory method is not necessary.

Conclusion: The method is considered valid for use as enforcement method in animal commodities with an LOQ of 0.01 mg/kg for isoxaflutole and IFT-DKN.

HPLC-UV method AR 109-95

HPLC-UV method AR 109-95 is intended as enforcement method for the determination of isoxaflutole, its diketonitrile metabolite IFT-DKN and its benzoic acid metabolite IFT-BA in animal commodities (fat, kidney, milk and eggs) [Guillet, et al., 1995, M-213160-01-1; Le Gren, 1995a/b/c, M-213162-01-1, M-262409-01-1, M-213164-01-1].

Milk residues were extracted by macerating with a mixture of 0.1% trifluoroacetic acid acidified water/ACN. The extract was purified using an Octyl cartridge, IFT and IFT-DKN being eluted in the same fraction.

Fat residues were extracted by macerating with a mixture of 0.1% trifluoroacetic acid acidified water/ACN. The extract was purified using an Octyl cartridge followed by a Diol cartridge.

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IFT-BA was eluted directly on the Octyl cartridge and IFT and IFT-DKN were eluted consecutively on the Diol cartridge.

Kidney residues were extracted by macerating with ACN. The extract was purified using an Octyl cartridge for IFT-DKN and additionally using a Diol cartridge for IFT. For determination of IFT-BA, the extract was purified using a Diol cartridge first followed by the purification using an Rdx cartridge.

Egg residues were extracted by macerating with ACN. The extract containing IFT-DKN was purified using an Octyl cartridge followed by a Diol cartridge for IFT-DKN. Attempts to analyse IFT in eggs were not successful, even with acidified extraction solvent, because IFT rapidly degrades onto IFT-DKN in presence of a basic matrix like eggs (pH around 9).

Quantification was carried out by HPLC-UV on a C18 column at 270 or 300 nm. Quantification of residues was done by external calibration using standards in solvent. Analyte levels were expressed as the corresponding analyte.

HPLC-UV Method AR 109-95 was validated according to a reduced validation scheme for bovine fat, bovine kidney, cow milk and hen eggs [Guillet, et al., 1995, M-213160-01-1; Le Gren, 1995a/b/c, M-213162-01-1, M-262409-01-1, M-213164-01-1]. Mixed analyte solutions were used for fortification of samples. Average recoveries at 0.01 mg/kg (milk), 0.02 mg/kg (eggs, tissues) and 0.1 mg/kg were within 70–120% limits and RSDs were within 20% for each analyte. Linearity was observed in the range of 10–1000 ug/L for IFT and IFT-DKN and in the range 25–1000 ug/L for IFT-BA. The equivalent concentration in the samples was not stated. The coefficient of correlation was r> 0.99 using 4–7 duplicate solvent standards. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg in milk, 0.006 mg/kg in eggs, fat and kidney) were detected in any of the control extracts. The valid LOQ was 0.01 mg/kg in milk (IFT, IFT-DKN), 0.02 mg/kg in eggs (IFT-DKN) and 0.02 mg/kg in fat and kidney (IFT, IFT-DKN and IFT-BA).

Conclusion: HPLC-UV method AR 109-95 is considered insufficiently validated for use as enforcement method, since a reduced validation scheme is used (n=3/level), a confirmation method is not available and no validation is available for muscle/meat and liver.

HPLC-UV method RPAC #45532

HPLC-UV method RPAC #45532, version 2.0, 4 March 1998, is intended as enforcement method for the determination of IFT, IFT-DKN and RPA 205834 in animal commodities (milk, eggs and tissues) [Lopes et al., 1998, M-166734-01-2].

Milk (50 g) was extracted by macerating with a mixture of 0.1% aqueous trifluoroacetic acid and ACN (5:18 v/v). The extract was purified using a C-8 cartridge column. The cartridge is eluted subsequently with water/ACN (90:10, v/v) and water/ACN (50:50, v/v). The first fraction contains IFT-BA and this fraction is discarded. Isoxaflutole, IFT-DKN and RPA 205834 were eluted and collected together in the second fraction.

Eggs were extracted with ACN. The extract was purified using a C-8 cartridge column, IFT-DKN being collected in the second fraction.

Tissues were analysed by a common moiety technique. Kidney, liver and muscle (10 g) were extracted using 0.1% aqueous trifluoroacetic acid. For beef fat or chicken skin with adhering fat (10 g) the extraction solvent was a 2:1 mixture of hexane and water. The fat hexane extracts were partitioned against an 18:5 mixture of ACN and water. The ACN-water phase was added to the aqueous extract and evaporated to remove all ACN. Isoxaflutole was converted to IFT-DKN by treatment of the extract with 10% NaOH. Trace enrichment/purification was obtained using a C-18 cartridge. IFT-DKN was eluted from the C-18 cartridge and collected in the second fraction.

All residue analyses were accomplished by HPLC-UV on a C-8 column at 270 or 300 nm. Quantification of residues was done by external calibration using standards in solvent. Analyte levels in milk and eggs were expressed as the corresponding analyte. For the animal tissue analysis the isoxaflutole related residues were determined as IFT-DKN but were reported as isoxaflutole. The

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molecular weight of isoxaflutole and IFT-DKN are the same, therefore, a correction factor for differences in molecular weight is not necessary.

HPLC-UV method RPAC #44882 is identical to HPLC-UV method RPAC #45532 for milk, except that fraction 1, containing IFT-BA, was analysed as well. HPLC-UV method RPAC #44882 was validated according to a full validation scheme for cow milk for IFT, IFT-DKN, RPA 205834 and IFT-BA [Shaffer, 1995, M-192304-01-2]. Two trials were required to optimize the method. Trial 1 was not successful due to low and highly variable recoveries of IFT-DKN. The retention times shifted significantly when the pH of the final solution was changed from 2.13 to 2.07. A second milk trial, incorporating the developer’s suggestions and taking care to mix the sample extract well, was successful. Mixed analyte solutions were used for the fortification of milk. Average recoveries at 0.02 and 2.0 mg/kg were within 70–120% limits and RSDs were within 20% for each analyte and matrix tested. Linearity (coefficient of correlation r> 0.9999) was observed in the range of 20–100 ng/mL using five single or duplicate standards in solvent. Equivalent concentrations in the samples were not stated. No interfering peaks > 0.3 LOQ (i.e. 0.006 mg/kg) were detected in any of the control samples. The valid LOQ was 0.02 mg/kg for IFT, IFT-DKN, RPA 205834 and IFT-BA.

HPLC-UV method RPAC #44933 (identical to HPLC-UV method RPAC #45532 for eggs) was validated according to a full validation scheme for IFT-DKN in hen eggs [Tew, 1995b, M-192317-01-1, Lowder, 1996, M-192281-01-2-01]. Average recoveries at 0.05 mg/kg (n=8) and 0.25 mg/kg (n=6) were within 70–120% limits and RSDs were within 20% for IFT-DKN in eggs. Linearity (coefficient of determination R2> 0.999) was observed in the range of 20–200 ng/mL using six single standards in solvent. Equivalent concentrations in the samples were not stated. No interfering peaks > LOQ (i.e. 0.05 mg/kg) were detected in any of the control extracts (n> 2). The valid LOQ was 0.05 mg/kg in eggs for IFT-DKN.

HPLC-UV method RPAC #45532 was validated by an independent laboratory according to a full validation scheme for milk, eggs, meat and kidney [Kaune, 2002, M-213302-01-1]. Validation was conducted for IFT, IFT-DKN and RPA 205834 in milk, IFT-DKN in eggs and IFT, IFT-DKN in meat and kidney. Animal origin for the tissues is not indicated for these samples. Separate solutions for each individual analyte were used for fortification of eggs and tissues. Mixed analyte solutions were used for fortification of milk. Average recoveries at 0.01–0.1 mg/kg for milk and eggs or 0.2–2.0 mg/kg for tissues were within 70–120% limits and RSDs were within 20% for each analyte and matrix tested. The calibration curve was not linear: a curve of the type y = a + bx + cx2 was applicable over the tested range of 5 to 50 ng (0.05 to 0.5 mg/L) using 4–5 single or duplicate standards in solvent. Equivalent concentration in the samples was not stated. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts. The valid LOQ was 0.01 mg/kg in milk (IFT, IFT-DKN and RPA 205834), 0.01 mg/kg in eggs (IFT-DKN), 0.2 mg/kg in meat and kidney (IFT, IFT-DKN).

Conclusion: HPLC-UV method RPAC #45532 is considered insufficiently validated for use as enforcement method, since a confirmation method is not available, the method for tissues is a common moiety method and no validation is available for fat and liver.

HPLC-UV method RPAC #45532 is considered valid for use as pre-registration analytical method in the range 0.02–2.0 mg/kg IFT, IFT-DKN, RPA 205834 and IFT-BA in milk and at 0.05–0.25 mg/kg IFT-DKN in eggs when using a linear calibration curve. The LOQ may be lowered to 0.01 mg/kg in milk and eggs when using a second order calibration curve.

HPLC-UV method RPAC #45532 is considered not suitable for use as pre-registration analytical method since the residues are analysed as a common moiety.

Analytical methods used in study reports in plant commodities

GC-MS method 00473 and its modifications

GC-MS method 00473, version January 1995, code HWI 6224-215, is a common moiety method for the determination of isoxaflutole related residues (sum of IFT, IFT-DKN and IFT-BA) in plant

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commodities [Nandihalli, 1995, M-020184-01-1]. Homogenised samples (25 g) were extracted by maceration with MeOH (3× 125 mL). Combined extracts were filtered. After addition of 2% NaOH solution isoxaflutole was hydrolysed to IFT-DKN in 1 hr at room temperature. The MeOH was evaporated off and 10 mL saturated NaCl was added to the low volume aqueous phase. The aqueous phase was washed subsequently with DCM and petroleum ether. Acidification (12 M HCl, pH 1.0) of the aqueous phase then allowed liquid-liquid partition of both IFT-DKN and IFT-BA into DCM (3×). The DCM phase was drained through anhydrous Na2SO4 and evaporated to dryness. The residue was re-dissolved in 1 M methanolic NaOH solution and heated for 1 hr at 100 ºC, whereby residues of IFT-DKN were hydrolysed to IFT-BA. Acidification (12 M HCl, pH 1.0) of the aqueous phase then allowed liquid-liquid partition of IFT-BA into DCM (3×). The DCM phase was drained through anhydrous Na2SO4 and evaporated to dryness. The residue was redissolved in DCM and IFT-BA was methylated to RPA 204497 using diazomethane (1 hr 30 ºC). Excess diazomethane was destroyed by addition of HAc. Residues of the ester derivative RPA 204497 are detected by GC-MS at m/z 251 (quantification) and m/z 267 (confirmation). Residues are quantified as RPA 204497, but are expressed as isoxaflutole equivalents in the trials.

GC-MS method 00473 was used in maize storage stability studies [Nandihalli, 1996, M-089371-01-1]. GC-MS method 00473 was validated according to a full validation scheme for maize forage, maize silage, maize grain and maize fodder [Nandihalli, 1995, M-020184-01-1]. Samples were fortified separately with IFT, IFT-DKN or IFT-BA. Average recoveries at 0.01 and 0.05 mg/kg (n=6 each) were within 70–120% limits, except IFT-BA in maize grain (0.01/0.05 mg/kg, average 67%/64%, n=6 each). Individual recoveries and RSDs were not reported. Additional recovery data from the storage stability study indicated that average recoveries at 0.2 mg/kg in maize silage and maize fodder and at 0.1 mg/kg in maize grain were within 70–120% limits (n=12, each analyte, each matrix) and RSD at this level was within 20%. Linearity (coefficient of correlation r> 0.9999) was observed in the range of 0.01–0.5 mg/L using six single solvent standards. The equivalent concentration in the samples was not stated. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts. The valid LOQ is 0.01 mg/kg for isoxaflutole related residues, expressed as isoxaflutole.

GC-MS-MS modification A of method 00473 was used in the Australian sugarcane supervised residue trials & storage stability studies (dated April 1999) [Davis and Keats, 1999a/b/c/d/e/f, M-284383-01-1, M-284347-01-1, M-284356-01-1, M-284369-01-1, M-284408-01-1 M-284324-01-1]. Modifications involved changes in sample types, extraction volumes, conditions for hydrolysis, diazomethane methylation and MS conditions. Homogenised samples of sugarcane billets (25 g) or sugarcane forage (25 g) were extracted with 2× 80 mL MeOH. Further sample preparation was identical to the original method except that an aliquot equivalent to 5 g sample matrix was taken for further analysis. The hydrolysis of IFT-DKN to IFT-BA was conducted at a lower temperature of 60 ºC. Diazomethane was directly added to the last DCM phase and IFT-BA was methylated to RPA 204497 (30 min, room temperature). Excess diazomethane was destroyed by addition of HAc and toluene was added to the extract. The extract was then reduced to the toluene phase. The toluene phase was filtered using a 0.45 μm PTFE filter and clean-up on a Florisil cartridge. The analytes were eluted with a toluene/acetone mixture (90:10, v/v). Residues of the ester derivative of RPA 204497 are detected by GC-MS-MS at m/z 251 and m/z 252 (parent ions as well as quantification ions).

GC-MS-MS modification A of method 00473 was partially validated for sugarcane billets and sugarcane forage [Davis and Keats, 1999a/b/c/d/e/f, M-284383-01-1, M-284347-01-1, M-284356-01-1, M-284369-01-1, M-284408-01-1 M-284324-01-1]. Samples were fortified separately with IFT, IFT-DKN or IFT-BA. Average recoveries at 0.01 mg/kg (n=5) for each compound were within 70–120% limits. Additional recovery data from the storage stability study indicated that average recoveries at 0.1 mg/kg in maize silage and maize fodder and at 0.1 mg/kg in maize grain were within 70–120% limits (n=12, each analyte, each matrix) and RSD at this level was within 20%. Recoveries at 10× LOQ were not verified. However, since residue levels in the trials were < LOQ, this is considered acceptable. Linearity (coefficient of correlation r> 0.9999) was observed in the range of 0.01–0.1 mg/L using three duplicate solvent standards. The equivalent concentration in the samples

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was not stated. No interfering peaks > LOQ were detected in any of the control extracts (n> 2, each matrix). The valid LOQ is 0.01 mg/kg for isoxaflutole related residues, expressed as isoxaflutole.

GC-MS-MS modification B of method 00473 was used in the 1999 chickpea supervised residue trials & storage stability studies (dated December–February 2000) [Lynch and Keats, 1999a/b, M-432364-01-1, M-432374-01-1, Lynch and Keats, 2000a/b/c, M-432434-01-1, M-432441-01-1, M-432444-01-1]. Modifications involved changes in sample types, extraction volumes, conditions for hydrolysis, diazomethane methylation and MS conditions. Homogenised chickpea seeds (25 g), chickpea forage (25 g) or chickpea straw (10 g) were extracted with 2× 100 mL MeOH. Further sample preparation was identical to the original method except that an aliquot equivalent to 5 g sample matrix was taken for further analysis and aqueous phases were washed with diethyl ether instead of petroleum ether. The hydrolysis of IFT-DKN to IFT-BA was conducted at a lower temperature of 60 ºC. The last DCM phase was drained through anhydrous Na2SO4. After addition of diazomethane IFT-BA was methylated to RPA 204497 (30 min, room temperature). Excess diazomethane was removed by nitrogen at 30–35 ºC and ACN was added to the extract. The extract was then reduced to 1 mL of ACN, made up to 5 mL with ACN and filtered using a 0.45 μm PTFE filter cartridge. Residues of the ester derivative of RPA 204497 are detected by GC-MS-MS at m/z 251 (parent ion) and m/z 189 and m/z 221 (quantification ions).

GC-MS-MS modification B of method 00473 was validated for chickpea seeds, chickpea forage and chickpea straw [Lynch and Keats, 1999a/b, M-432364-01-1, M-432374-01-1, Lynch and Keats, 2000a/b/c, M-432434-01-1, M-432441-01-1, M-432444-01-1]. Samples were fortified separately with IFT, IFT-DKN or IFT-BA. Average recoveries at 0.01 and 0.1 mg/kg (n=3 or 6) for each compound were within 70–120% limits. Individual recoveries and RSDs were not reported. Linearity was not observed: a second order calibration curve (correlation coefficient r> 0.99) was fitted to the data in the range 0.005–0.5 mg/L using six triplicate solvent standards. The equivalent concentration in the samples was not stated. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts (n> 2, each matrix). The valid LOQ is 0.01 mg/kg for isoxaflutole related residues, expressed as isoxaflutole.

GC-MS modification C of method 00473 was used in the Brazilian sugarcane supervised residue trials (dated March 2000) [Tornisielo, 2000a/b, M-287038-01-1, M-287041-01-1]. Modifications involved changes in sample types, extraction volumes and diazomethane methylation. Homogenised sugarcane billets (20 g) were extracted by maceration with 2× 95 mL MeOH. Further sample preparation was identical to the original method. The last DCM phase was reduced to 5 mL. After addition of diazomethane, IFT-BA was methylated to RPA 204497 (time and temperature not stated) and the sample was evaporated to dryness. The residue was redissolved in hexane.

GC-MS modification C of method 00473 was validated for sugarcane billets [Tornisielo, 2000a/b, M-287038-01-1, M-287041-01-1]. Samples were fortified with IFT only. Average recoveries at 0.01 and 0.02 mg/kg (n=3, each) were within 70–120% limit and RSD was within 20% at these levels. Recoveries at 10× LOQ were not verified. However, since residue levels in the trials were < LOQ, this is considered acceptable. Linearity (coefficient of determination R2> 0.999) was observed in the range of 0.02–0.15 ng/mL using five single solvent standards. The equivalent concentration in the samples was not stated. No interfering peaks > LOQ were detected in any of the control extracts (n=2). The valid LOQ is 0.01 mg/kg for isoxaflutole related residues, expressed as isoxaflutole.

GC-MS modification D of method 00473 was used in the Mexican sugarcane supervised residue trials (dated November 2000) [Gough, 2000, M-238729-01-1]. Modifications involved changes in sample types and diazomethane methylation. Homogenised samples of sugarcane billets (12.5 g) were extracted with 3× 125 mL MeOH (3× 125 mL). The last DCM phase was drained through anhydrous Na2SO4. After addition of diazomethane, IFT-BA was methylated to RPA 204497 (30 min, room temperature) and the sample was evaporated to dryness. The residue was redissolved in DCM and cleaned-up on a Florisil cartridge. The analytes were eluted with an EtOAc/DCM (1:1, v/v). The eluent was evaporated to dryness and redissolved in DCM.

GC-MS modification D of method 00473 was validated for sugarcane billets [Gough, 2000, M-238729-01-1]. Samples were fortified separately with IFT, IFT-DKN or IFT-BA. Only limited

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validation results are available. Average recoveries at 0.01, 0.02, 0.05, 0.1 mg/kg analyte were within 70–120% limits (n=1–4 each), except for IFT at 0.01 mg/kg (128%, n=2) and IFT-DKN at 0.01 mg/kg (122%, n=2). The RSD was within 20% at 0.02 mg/kg IFT and at 0.01 mg/kg IFT-DKN or IFT-BA; the RSD was not verified at other levels. This is considered acceptable, since the residue levels in the trials are at LOQ. Linearity was not verified, since this is covered by the original method. No interfering peaks > LOQ (i.e. 0.01 mg/kg) were detected in any of the control extracts (n> 2). The valid LOQ is 0.01 mg/kg for isoxaflutole related residues, expressed as isoxaflutole.

Overall, GC-MS method 00473 and its modifications are considered fit for purpose:

� The original method is considered valid at 0.01–0.2 mg/kg total residues in maize forage, maize grain and maize fodder.

� Modification A is considered valid at 0.01 mg/kg total residues in sugarcane billets and sugarcane forage.

� Modification B is considered valid at 0.01–0.1 mg/kg total residues in dry chickpea seeds, chickpea forage and chickpea straw. However, since residue values for chickpea forage in the supervised trials ranged from < 0.01–0.19 mg/kg, validation at higher levels is desirable.

� Modification C is considered valid at 0.01–0.02 mg/kg total residues in sugarcane billets. � Modification D is considered valid at 0.01–0.02 mg/kg total residues in sugarcane billets.

HPLC-MS-MS method CAL#19-03 and its modifications

HPLC-MS-MS method CAL#019-03, revision 1, issued 29 December 1998, allows the individual determination of IFT and its metabolites IFT-DKN and IFT-BA in plant commodities [Wickremesinhe, 1998a, M-286215-01-1]. Revision 1 differs from the original version (issued on August 18, 1998) in that it includes the results from the validation study [Wickremesinhe, 1998b, M-240820-01-1]. Homogenised samples (10 g) were mixed with 10 mL 1% formic acid and left for 5–10 min (to hydrate dry matrices and to stabilize isoxaflutole). The sample mixture was extracted with 90 mL MeOH and homogenised. The filtered extract was acidified (1 M HCl) and 10% NaCl was added. The extract was purified by liquid-liquid partitioning into methylene chloride. After acidification with 1% formic acid, the methylene chloride phase was evaporated to the aqueous remainder. The residue was diluted with 1% formic acid and further purified by solid-phase extraction (RP 102 column) using 40–70% ACN in water. Two fractions were collected: fraction 1 contained IFT-DKN and IFT-BA and fraction 2 contained IFT. Fractions were adjusted to 100 mL with ACN and water to contain 40% ACN in water. Quantification was performed by HPLC-MS-MS analysis. A turbo-ion interface (atmospheric pressure ionization) was used for the analysis of fraction 1 (with IFT-DKN and IFT-BA), whereas a heated nebulizer interface was used for the analysis of fraction 2 (with IFT, to avoid matrix suppression). The mass transitions monitored were m/z 358 → 79 for IFT-DKN and m/z 267 → 159 for IFT-BA (fraction 1) and m/z 358 → 79 for IFT (fraction 2). For all analytes quantification was done using external calibration with standards in solvent.

HPLC-MS-MS method CAL#019-03, revision 1, was used in the 1998 supervised residue trials on maize in the US [Mickelson, 1999a, M-240663-01-1]. HPLC-MS-MS method CAL#019-03, was validated according to the full validation scheme for maize grains, maize flour, wheat straw and mustard leaves [Wickremesinhe, 1998b, M-240820-01-1]. Samples were fortified with a mixture of IFT, IFT-DKN or IFT-BA. Average recoveries at 0.01 and 0.10 mg/kg (n=5 each) were within 70–120% limits and RSDs were within 20% for each analyte and matrix. An exception is wheat straw at 0.10 mg/kg IFT-BA with an average recovery of 65% (n=5, range 62–72%). Additional limited validation data are available in the supervised residue trials [Mickelson, 1999a, M-240663-01-1]. Average recoveries at 0.01, 0.1 and 10 mg/kg (n=2 each) in maize forage and maize fodder were within 70–120% limits for each analyte. Linearity (correlation coefficient r> 0.99) was observed in the range of 0.5–5 ng/mL using four duplicate standards in solvent. A 0.5–5 ng/mL standard is equivalent to 0.005–0.05 mg/kg in the sample. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control samples. The valid LOQ is 0.01 mg/kg for each analyte. After validation, the validation results were included in the method description and the method became revision 1.

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Modification A of HPLC-MS-MS method CAL#019-03 was used in the field rotational crop study for matrices with low moisture content (fodder, hay, straw and grains) [Mickelson, 2000, M-240710-01-1]. Modifications involved changes in extraction. Low moisture samples (5 g) were extracted with 20 mL 1% formic acid and 100 mL MeOH (20:100, v/v). The extract was cleaned up as described in revision 1 of the method. Final fractions were adjusted to 50 mL instead of 100 mL. Only limited concurrent recovery data (n=1/level, at 0.01–0.05–0.5 mg/kg, within 70–120%, all analytes) are available for wheat hay, wheat grains, wheat straw, soya bean seeds, soya bean hay, sorghum grains and sorghum fodder. Recoveries are not satisfactory for IFT-BA in wheat hay (62%, n=1, 0.01 mg/kg), wheat straw (68%, n=1, 0.01 mg/kg) and soya bean seeds (64%, n=1, 0.01 mg/kg). No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control samples.

Modification B of HPLC-MS-MS method CAL#019-03 was used in the field rotational crop study on fresh commodities [Mickelson, 2000, M-240710-01-1]. Modifications involved only minor changes in the RP102 clean-up part of the method. Only limited concurrent recovery data are available for mustard leaves, turnip roots, turnip tops, wheat forage, soya bean forage, radish roots, radish tops, sorghum forage (at n=1/level, 0.01–0.05–0.5 mg/kg, within 70–120%, all analytes). Recoveries are not satisfactory for IFT-BA in turnip tops (69%, n=1, 0.01 mg/kg) and wheat forage (69%, n=1, 0.01 mg/kg). Linearity (coefficient of correlation r> 0.99) was observed in the range of 0.5–5 ng/mL using four single standards in solvent. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control samples.

Modification C of HPLC-MS-MS method CAL#019-03 was used in the 2005 and 2006 Canadian and US supervised field trials on maize [Fischer and Helfrich, 2007, M-285889-01-1; Vaughn and Cosgrove, 2007, M-286216-02-1]. Modifications involved changes in extraction, addition of internal standards and omission of the RP 102 clean-up step and a different MS system. Homogenised samples (5 g) were extracted with 10 mL 1% formic acid and 90 mL MeOH. Mixed internal standards (13C6-IFT, 13C6-IFT-DKN, 13C6-IFT-BA, and 0.025 mg/L each) were added together with the HCl solution. After liquid-liquid partition, the DCM phase was acidified with 1% formic acid and evaporated to 1 mL. The sample was diluted with 1% aqueous formic acid solution and ACN (50:50 or 60:40, v/v, 100 mL). A 0.1 mL aliquot was used for injection. HPLC-MS-MS with electrospray ionization was used for the analysis of IFT, IFT-DKN and IFT-BA. The mass transitions monitored were m/z 358 → 79 for IFT, m/z 358 → 79 for IFT-DKN and m/z 267 → 159 for IFT-BA as well as the mass transitions for their 13C isotopes.

Modification C of HPLC-MS-MS method CAL#019-03 was validated according to a reduced scheme for maize forage, maize grains and maize fodder [Fischer and Helfrich, 2007, M-285889-01-1]. Samples were fortified separately with IFT, IFT-DKN or IFT-BA. Average recoveries at 0.01 mg/kg (n=14–22) and 0.20 mg/kg (n=3, IFT) or 0.27 mg/kg (n=3, IFT-DKN, IFT-BA) were within 70–120% limits for each analyte. The RSD was within 20%, except for IFT-DKN and IFT-BA at 0.01 mg/kg in maize forage (24%, 21%) and for IFT-BA at 0.01 mg/kg in maize fodder (27%). Linearity (1/× weighted, correlation coefficient r> 0.99) was observed in the range of 0.002–2.0 mg/kg using seven duplicate standards in solvent with internal standards. No interfering peaks > LOD (i.e. 0.003 mg/kg IFT, 0.005–0.007 mg/kg IFT-DKN, 0.006–0.01 mg/kg IFT-BA) were detected in any of the control extracts (n> 2), except 0.007 mg/kg IFT-BA in maize grain. Based on these results, the valid LOQ is 0.01 mg/kg for IFT, 0.02 mg/kg for IFT-DKN and 0.03 mg/kg for IFT-BA.

Conclusion: HPLC-MS-MS method CAL#19-03 is generally insufficiently validated. Modification C needs adaption of LOQs to 0.02 mg/kg for IFT-DKN and 0.03 mg/kg for IFT-BA. Additional validation data are needed for commodities analysed in the field rotational crop study:

� Revision 1 is considered valid at 0.01–0.10 mg/kg IFT, IFT-DKN and IFT-BA in maize grains, maize flour, mustard leaves, maize forage and maize fodder. The method is not valid in wheat straw. Since IFT-BA levels in the supervised trials on maize forage were in the range < 0.01–0.15 mg/kg, validation at higher levels is desirable.

� Modification A is considered insufficiently validated. Additional validation data for the commodities analysed in the field rotational crop study are needed.

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� Modification B is considered insufficiently validated. Additional validation data for the commodities analysed in the field rotational crop study are needed.

� Modification C is considered valid at 0.01–0.2 mg/kg IFT, 0.02–0.3 mg/kg IFT-DKN and 0.03–0.3 mg/kg IFT-BA in maize forage, maize grains and maize fodder. Results below 0.02 mg/kg IFT-DKN and below 0.03 mg/kg IFT-BA in the supervised field trials need to be reported as < 0.02 mg/kg IFT-DKN and < 0.03 mg/kg IFT-BA.

HPLC-MS-MS method 00985/M001

HPLC-MS-MS method 00985/M001, version 13 November 2006, was developed to determine individual residues of IFT and its metabolites IFT-DKN and IFT-BA in plant materials [Schoening and Wolters, 2006, M-280607-01-1]. Samples (10 g) were extracted with MeOH/1% formic acid (90:10, v/v). After filtering, the corresponding 13C6 labelled analytes were added. The solution was made up to volume with water (pH 3, using formic acid) and MeOH. An aliquot of the extract was 5× diluted with water/ACN (90/10, v/v, pH 3 using formic acid), filtered and subjected to HPLC-MS-MS analysis with electrospray ionisation. For quantification external calibration with standard solutions was used quoting the peak area ratio of the analytes and the corresponding isotopically labelled internal standards. Quantification was carried out by monitoring the ion transitions m/z 360/366 → 251/257 (IFT), m/z 358/364 → 79/79 (IFT-DKN) and m/z 267/273 → 159/165 (IFT-BA).

HPLC-MS-MS method 00985/M001 was used in the EU maize and poppy seed trials [Zimmer and Wieland, 2007, M-282674-01-1; Wolters and Erler, 2007a/b, M-284416-01-1, M-284423-01-1; Wolters, 2007a/b, M-285005-01-1, M-285014-01-1; Zimmer, 2006, M-281611-01-1; Billian and Reinecke, 2010, M-360144-02-1; Billian and Krusell, 2010a/b, M-364166-01-1, M-360114-02-1; Noss and Reinecke, 2010, M-366025-01-1]. HPLC-MS-MS method 00985/M001 was validated according to the full validation scheme for maize grains, maize forage and sweet corn (cobs without husks) [Schoening and Wolters, 2006, M-280607-01-1] and poppy seeds [Billian and Reinecke, 2010, M-360144-02-1]. Grinding of poppy seeds led to residues about a factor 2 higher compared to non-grinded seeds. Therefore, poppy seeds need to be grinded prior to extraction (e.g. 30 sec using an IKA A11 basic mixer or pestling by hand). Samples were fortified with a mixture of IFT, IFT-DKN or IFT-BA. Average recoveries at 0.01 mg/kg and 0.10 mg/kg were within 70–120% limits and RSDs were within 20% for each analyte and matrix. Linearity (coefficient of correlation r > 0.99 for 1/× weighted) was observed in the range of 0.01–50 ng/mL standards using nine single solvent standards and internal standards at 1 ng/mL. Equivalent concentrations in the samples were not stated. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts. The valid LOQ is 0.01 mg/kg for each analyte in all matrices tested.

Conclusion: HPLC-MS-MS method 00985/M001 is considered valid at 0.01–0.10 mg/kg IFT, IFT-DKN and IFT-BA in maize grains, maize forage, sweet corn (cobs without husks) and poppy seeds (which need to be grinded prior to extraction). Additional validation data are needed for maize fodder. Since IFT and IFT-DKN levels in the supervised residue trials on maize forage ranged from < 0.01–13 mg/kg and < 0.01–2.6 mg/kg, respectively, validation at higher levels is desirable.

HPLC-MS-MS method IS-004-P10 and its modifications

HPLC-MS-MS method IS-004-P10-01, issued 18 February 2010, was developed for the analysis of IFT and IFT-DKN in plant matrices [Stoughton, 2010a, M368825-01-1]. Homogenized samples (5 g) were extracted by blending with a 1% aqueous formic acid/MeOH (5:20, v/v) solution. The extraction was repeated with MeOH. The extract was filtered using a Bond Elute cartridge. An isotopic internal standard mixture (13C6-IFT, 13C6-IFT-DKN, 1 mg/L each) was added to the extract. An aliquot of the sample was then diluted 1:1 with 0.1% formic acid in water. The sample was analysed by HPLC-MS-MS for IFT and IFT-DKN. Quantification was based on the use of internal standards and comparisons of peak areas with those of known standards. The detection by MS-MS was performed with a turbo ion spray interface operated in positive ion mode and multiple reaction monitoring (MRM): m/z 360 to 251 for IFT and m/z 358 to 79 for IFT-DKN.

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HPLC-MS-MS method IS-004-P10-01 was used in soya bean residue trials [Dallstream and Fisher, 2010, M-368661-01-1; Beedle and Dallstream, 2010, M-368669-01-1]. HPLC-MS-MS method IS-004-P10-01 was validated according to the full validation scheme for soya bean seed [Stoughton, 2010c, M-368828-01-1]. Samples were fortified with a mixture of IFT and IFT-DKN. Average recoveries at 0.01 mg/kg and 0.10 mg/kg were within 70–120% limits and RSDs were within 20% for each analyte in soya bean seeds. Linearity (coefficient of determination R2> 0.99) was observed in the range of 0.12–9.0 ng/mL using seven duplicate solvent standards. Equivalent concentrations in the samples were 0.0024–0.180 mg/kg. No interfering peaks > 0. 3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts. The valid LOQ is 0.01 mg/kg for each analyte. On completion of the validation study, method IS-004-P10-01 was updated to include the confirmatory MRM transitions m/z 358 to 64 for IFT and m/z 358 to 64 for IFT-DKN. The updated method was issued and assigned a method number of IS-004-P10-02 (dated 12 May 2010) [Stoughton 2010b, M368826-01-1].

Modification A of HPLC-MS-MS method IS-004-P10-01 was used in soya bean processing studies [Fischer, 2010, M-368662-01-1]. The modification involved changes in sample type and sample weight. Samples of 5 g soya bean seed, soya bean meal, soya bean hulls, soya bean oil, soymilk or 1.0 g for soya bean aspirated grain fractions were homogenised before extraction.

Modification A of HPLC-MS-MS method IS-004-P10-01 was validated according to a reduced validation scheme for soya bean seed, soya bean meal, soya bean hulls, soya bean oil, soymilk and soya bean aspirated grain fractions [Fischer, 2010, M-368662-01-1]. Samples were fortified with a mixture of IFT and IFT-DKN. Average recoveries at 0.01 mg/kg (n=3) and 0.10 mg/kg (n=3) or 0.5 mg/kg (n=2, aspirated grain fractions) were within 70–120% limits and RSDs were within 20% for each analyte and matrix. Linearity (correlation coefficient r> 0.99) was observed in the range of 0.0–9.0 ng/mL using seven duplicate solvent standards. Equivalent concentrations in the samples were 0.0–0.180 mg/kg. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control extracts. The valid LOQ is 0.01 mg/kg for each analyte and matrix.

Modification B of HPLC-MS-MS method IS-004-P10-01 was used in storage stability studies [Klasmeier, 2012, M-442915-01-1]. The modification involved changes in extraction and addition of filtration steps. Homogenized samples (5 g) were extracted by blending once with a 1% aqueous formic acid/MeOH (10:40, v/v) solution. The extract was filtered on glass wool. Just prior to analysis the sample was filtered with 0.45 um Nylon filter. The detection by MS-MS was performed with a turbo ion spray interface operated in positive ion mode at m/z 360.1 to 250.9 and 219.8 for isoxaflutole and in negative ion mode at m/z 358.0 to 79.0 and 64.0 for IFT-DKN for quantification and confirmation.

Modification B of HPLC-MS-MS IS-004-P10-01 was partially validated for oranges, soya bean seeds, dry pinto beans and sugarcane billets [Klasmeier, 2012, M-442915-01-1]. Samples were fortified separately with isoxaflutole or IFT-DKN. Average recoveries at 0.1 mg/kg (n=3) were within 70–120% limits for each analyte. The RSD was within 20%. Recoveries at the LOQ were not verified, but this is considered acceptable for the storage stability studies at 0.1 mg/kg. Linearity (1/× weighted, correlation coefficient r> 0.99) was observed in the range of 0.12–9.0 ng/mL using five single standards in solvent with internal standards. No interfering peaks > LOD (i.e. 0.003 mg/kg were detected in any of the control extracts (n> 2)

Conclusion: HPLC-MS-MS method IS-004-P10 and its modifications are considered fit for purpose:

� The original method is considered valid at 0.01–0.1 mg/kg in dry soya bean seed. � Modification A is considered valid at 0.01–0.1 mg/kg in dry soya bean seed, soya bean meal,

soya bean hulls, soya bean oil, soymilk and 0.01–0.5 mg/kg in soya bean aspirated grain fractions.

� Modification B is considered valid at 0.1 mg/kg in oranges, dry soya bean seeds, dry pinto beans, and sugarcane billets.

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HPLC-MS-MS method ATM-0054.01

HPLC-MS-MS method ATM-0054.01, version August 2011, was developed to determine individual residues of IFT, IFT-DKN and IFT-BA in plant materials [Ellis, 2012, M-425905-02-1]. Homogenised samples (10 g) were extracted with 20:80 water:ACN. An aliquot of the extract was reduced to dryness and reconstituted in water acidified with 0.22% formic acid. The reconstituted sample was then filtered and subjected to analysis by HPLC-MS-MS. Quantitation was achieved using matrix matched analytical standards and 13C6-labelled internal standards for isoxaflutole, diketonitrile metabolite and the benzoic acid metabolite. The monitored ion transitions were m/z 360 → 251 for IFT, m/z 358 → 79 for IFT-DKN and m/z 267 → 159 for IFT-BA. The metabolites are not expressed as isoxaflutole equivalents.

HPLC-MS-MS method ATM-0054.01 was validated according to the full validation scheme for oat grain, lupin seed, and mung bean forage [Ellis, 2012, M-425905-02-1]. Samples were fortified with a mixture of IFT, IFT-DKN and IFT-BA. Average recoveries at 0.01 mg/kg and 1.0 mg/kg were within 70–120% limits and RSDs were within 20% for each analyte and matrix. Linearity (coefficient of correlation r> 0.999 for 1/× weighted) was observed in the range of 0.5–150 �g/L using five single matrix matched standards. Equivalent concentrations in the samples were not stated. No interfering peaks > 0.3 LOQ (i.e. 0.003 mg/kg) were detected in any of the control samples. The valid LOQ is 0.01 mg/kg for each analyte and matrix tested.

Modification A HPLC-MS-MS method ATM-0054.01 was used in the Australian chickpea trials [Ellis, 2012, M-425905-02-1]. The method modification involved using less sample material (5 g), increased extraction volumes and modified aliquots for further analysis.

Modification A was validated according to a full validation scheme for chickpea seeds and chickpea straw and a reduced validation scheme for chickpea forage [Ellis, 2012, M-425905-02-1]. Samples were fortified with a mixture of IFT, IFT-DKN or IFT-BA. Average recoveries at 0.01 mg/kg and 1.0 mg/kg were within 70–120% limits and RSDs were within 20% for each analyte and matrix. Linearity (coefficient of correlation r> 0.999 for 1/× weighted) was observed in the range of 0.5–150 ug/L using six single matrix matched standards. Equivalent concentrations in the samples were not stated. No interfering peaks were detected in any of the control samples (< 0.01 mg/kg for each analyte). The valid LOQ is 0.01 mg/kg for each analyte and matrix tested and 0.03 mg/kg for total residues.

Conclusion: HPLC-MS-MS method ATM-0054.01 and its modifications are considered fit for purpose:

� The original method is considered valid at 0.01–0.1 mg/kg in oat grain, lupin seed, and mung bean forage.

� Modification A is considered valid at 0.01–0.1 mg/kg in dry chickpea seeds, chickpea forage and chickpea straw. Since IFT-BA levels in the supervised trials on chickpea forage ranged from < 0.01–0.55 mg/kg and in chickpea straw ranged from < 0.01–0.12 mg/kg, validation at higher levels is desirable.

Analytical methods used in study reports in animal commodities


HPLC-UV method RPAC #44882, version 2.0, 27 October 1995, was developed to determine individual residues of IFT and its metabolites IFT-DKN, RPA 205834, and IFT-BA in milk [Tew, 1995a, M-192311-01-1]. The method was used in dairy cow feeding studies [Tew, 1995a, M-192311-01-1] and storage stability studies in milk [Lowder, 1996, M-192281-01-2-01]. The method is identical to HPLC-UV method RPAC #45532 for milk except that fraction 1, containing IFT-BA, is kept. Validation results for IFT, IFT-DKN, RPA 205834 and IFT-BA are available in the section describing method RPAC #45532.

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HPLC-UV method RPAC #44933, version 2.0, 17 November 1995 was developed to determine metabolite IFT-DKN in eggs [Tew, 1995b, M-192317-01-1]. The method is identical to HPLC-UV method RPAC #45532 for eggs and was used in laying hen feeding studies [Tew, 1995b, M-192317-01-1] and storage stability studies in eggs [Lowder, 1996, M-192281-01-2-01]. Validation results for IFT-DKN are available in the section describing method RPAC #45532.

HPLC-MS-MS method RPAC #44996 and its modifications

HPLC-MS-MS method RPAC #44996, version 1.0, 4 December 1995, was developed to determine RPA 207048 in animal tissues [Tew, 1995a/b, M-192311-01-1, M-192317-01-1, Lowder, 1996, M-192281-01-2-01]. Samples (25 g) were extracted twice with 0.1% aqueous trifluoroacetic acid and ACN (25:90, v/v). The combined filtered extracts were washed with hexane. A portion of the extract was reduced by evaporation and then diluted with NaCl solution. Residues are then partitioned into DCM and drained through anhydrous Na2SO4. The DCM phase was evaporated and the remaining residue was dissolved in water/ACN (80:20) prior to quantification by HPLC-MS-MS. Matrix matched standards were used to compensate for any matrix effects that may enhance or depress the analyte signal. The ion transitions monitored were 361/79 (RPA 207048).

HPLC-MS-MS method RPAC #44996, version 1.0 also provided good results for the parent compound and metabolites IFT-DKN and RPA 205834. Therefore the method was rewritten as HPLC-MS-MS method RPAC 44996, version 2.0, 29 January 1996, to incorporate these metabolites as well [Hudson et al., 1996, M-262714-01-1]. The ion transitions monitored were 358/79 (IFT), 361/79 (RPA 207048), 358/79 (IFT-DKN), and 360/79 (RPA 205834). During sample extraction and clean-up, parent IFT is partially degraded to IFT-DKN. Parent IFT is only stable at pH 2; metabolite RPA 207048 is unstable at pH 2. Since the method measures IFT and IFT-DKN, any degradation of IFT is measured as increased IFT-DKN, so that the entire amount of residue in the original sample is measured even though the character of the residue may have changed. For this reason, separate samples need to be prepared for verification of recovery for IFT, since residues will appear as IFT and its metabolite IFT-DKN.

HPLC-MS-MS method #44996 was used during the conduct of the dairy cow feeding studies, the laying hen feeding studies and storage stability studies in animal tissues [Tew, 1995a/b, M-192311-01-1, M-192317-01-1, Lowder, 1996, M-192281-01-2-01]. The method was validated by either fortifying IFT alone, or a mixture of IFT-DKN, RPA 207048, and RPA 205834. The recoveries for IFT were measured by summing up residues of IFT and IFT-DKN. Concurrent recovery results from the cow and hen feeding studies are summarized in the validation report [Hudson et al., 1996, M-262714-01-1]. Only limited recovery data (n=1–2/level, within 70–120%) are available for IFT, IFT-DKN, RPA 205834 and RPA 207048 in bovine liver (0.05, 0.25 and 2.0 mg/kg), bovine kidney (0.05, 0.45 and 0.60 mg/kg), bovine muscle (0.05 mg/kg only), bovine fat (0.05 mg/kg only), chicken liver (0.05, 0.45, 0.60 and 1.0 mg/kg), chicken muscle (0.05 mg/kg only) and chicken skin plus fat (0.05 and 0.45 mg/kg). An RSD within 20% was found for IFT in bovine liver at 0.05 mg/kg (n=3) and for IFT-DKN in chicken liver at 0.05 mg/kg (n=3). Additional recovery and precision data are available from the storage stability study. Average recoveries at 0.25 mg/kg for each analyte were within 70–120% limits and RSDs were within 20% for each analyte and matrix. Linearity (coefficient of correlation r> 0.99) was observed in the range of 0–150 ng/mL or 0–75 ng/mL using 6–7 single matrix matched standards. Equivalent concentrations in the samples were not stated. No interfering peaks were detected in any of the control samples (< 0.05 mg/kg for each analyte). The reported LOQ is 0.05 mg/kg for each analyte and matrix tested, but precision data at this level are not available (except in chicken and bovine liver).

Conclusion: HPLC-MS-MS method #44996 is considered suitable for determination of IFT, IFT-DKN, RPA 205834 and RPA 207048 in animal tissues. However, the method is considered insufficiently validated. Validation is sufficient at 0.25 mg/kg for each analyte and each tissue. Additional validation data are needed for muscle, kidney, and fat in order to establish precision at the

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reported LOQ of 0.05 mg/kg, since only 1–2 recoveries were available. Isoxaflutole is partly degraded because of extraction conditions, but is measured as an increased amount of IFT-DKN.

Since residue levels in the feeding studies ranged from < 0.05–0.503 mg/kg for IFT-DKN in bovine kidney, < 0.05–1.84 mg/kg for IFT-DKN in bovine liver, 0.123–0.645 mg/kg for IFT-DKN in hen liver, < 0.05–0.810 mg/kg for RPA 205834 mg/kg in bovine liver, additional validation at higher levels is desirable for kidney and liver.

Because the extraction solvent resembles the extraction solvent used in HPLC-UV method RPAC #45532 for milk and eggs, the method is possibly also suitable for milk and eggs; validation data on these commodities are desirable.

Stability of pesticide residues in stored analytical samples

The Meeting received information on storage stability of parent and its metabolites IFT-DKN and IFT-BA in plant commodities. In addition the Meeting received information on storage stability of parent and its metabolites IFT-DKN, RPA 205834 and RPA 207048 in animal commodities.

Study 1

Storage stability was investigated in maize commodities [Nandihalli, 1996, M-089371-01-1]. Homogenized untreated sample materials (25 g each) were independently spiked with IFT, IFT-DKN and IFT-BA. Fortification levels were 0.210 mg/kg for maize forage and maize fodder, 0.105 mg/kg for maize grain. The samples were stored at –10 °C or lower for 15 months. Samples were analysed in duplicate at various intervals using GC-MS method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 (and expressed as spiked analyte). Average concurrent recoveries at 0.21 mg/kg for maize silage and maize fodder and at 0.105 mg/kg for maize grain were within 70–120%. Levels in control samples were < 0.003 mg/kg (0.3 LOQ).

During the storage period the temperature increased to –5.5 to –3.9 ºC on two consecutive days, –0.4 to +2.0 ºC on two consecutive days and –8.2 to –2.2 ºC on four consecutive days. Since the samples remained frozen, this deviation did not significantly affect the results of the study. Storage stability results and concurrent recoveries are shown in Table 37. The presented results for the stored samples are not corrected by the concurrent (freshly spiked) recoveries.

Conclusion: The storage stability results indicate that there were no significant losses of total isoxaflutole residues (sum of IFT, IFT-DKN and IFT-BA) in maize grains, maize forage and maize fodder stored at –10 °C for at least 15 months. Any degradation from IFT to IFT-BA or from IFT-DKN to IFT-BA remains undetected, because of the common moiety method used.

Table 37 Storage stability of 0.1–0.2 mg/kg IFT, IFT-DKN or IFT-BA in frozen maize commodities at -10 ºC

Commodity Storage time (months)

IFT % remaining mean, n=2

IFT concurrent recovery mean, n=2

IFT-DKN % remaining mean, n=2

IFT-DKN concurrent recovery, mean, n=2

IFT-BA % remaining mean, n=2

IFT-BA concurrent recovery, mean, n=2

Maize forage

0 1 2 3 6 12 15

– 96 77 85 77 74 88

78 89 85 74 88 87 95

– 81 68 63 72 71 82

102 90 79 85 84 92 92

– 85 79 83 81 81 89

86 99 87 79 90 87 90

Maize fodder

0 1 2 3 6 12 15

– 84 78 77 71 75 87

97 73 86 83 81 88 91

– 64 60 61 63 66 74

78 82 70 74 75 72 90

– 83 79 73 77 71 83

92 95 85 78 86 78 90

Maize 0 – 79 – 77 – 79

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Commodity Storage time (months)







grain 1 2 3 6 12 15

90 74 83 62 78 77

100 82 85 85 92 89

70 67 74 64 75 77

71 76 78 72 84 86

77 61 68 62 59 62

85 74 67 80 69 75

Study 2

Storage stability was investigated in chickpea commodities [Lynch and Keats, 1999b, M-432374-01-1, Lynch and Keats, 2000a, M-432434-01-1]. Homogenized untreated sample materials of chickpea forage, chickpea seeds and chickpea straw were independently spiked with IFT, IFT-DKN and IFT-BA at a fortification level of 0.10 mg/kg. The samples were stored at –20 °C for 11 months. Samples were analysed in triplicate at various intervals using GC-MS-MS modification B of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 (and expressed as spiked analyte). Average concurrent recoveries at 0.01 mg/kg were within 70–120% for each analyte and matrix. Levels in control samples were < 0.003 mg/kg (0.3 LOQ).

Storage stability results and concurrent recoveries are shown in Table 38. Presented results for the stored samples are not corrected by the concurrent (freshly spiked) recoveries.

Conclusion: The storage stability results indicate that there were no significant losses of total isoxaflutole residues (sum of IFT, IFT-DKN and IFT-BA) in chickpea forage, chickpea seeds or chickpea straw stored at –20 °C for at least 11 months. Any degradation from IFT to IFT-BA or from IFT-DKN to IFT-BA remains undetected, because of the common moiety method used.

Table 38 Storage stability of 0.1 mg/kg IFT, IFT-DKN or IFT-BA in chickpea commodities at -20 ºC

Commodity Storage time



IFT-DKN %remaining, mean, n=3

IFT-DKN concurrent recovery mean, n=3



Chickpea forage

3 days 78 79 78 75 78 81

11 months 73 95 73 72 73 99 Chickpea grain

3 days 76 78 76 76 76 82

11 months 78 80 78 75 78 87 Chickpea straw

3 days 73 79 73 77 73 76

11 months 74 80 74 82 74 87

Study 3

Storage stability was investigated in sugarcane commodities [Davis and Keats, 1999e/f, M-284408-01-1, M-284324-01-1] Homogenised untreated samples of sugarcane billets and sugarcane forage were independently spiked with IFT, IFT-DKN and IFT-BA at a fortification level of 0.10 mg/kg eq. The samples were stored at –20 °C for a period of 20 months. Samples were analysed in duplicate at various intervals using GC-MS modification A of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and residues were expressed as isoxaflutole equivalents. Average concurrent recoveries at 0.01 mg/kg eq were within 70–120% for each analyte and matrix. Levels in control samples were < 0.01 mg/kg (LOQ).

Storage stability results and concurrent recoveries are shown in Table 39. The presented results for the stored samples are not corrected by the concurrent (freshly spiked) recoveries.

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Conclusion: The storage stability results indicate that there were no significant losses of total isoxaflutole residues (sum of IFT, RPA 202238 and IFT-BA) in sugarcane billets or sugarcane forage stored at –20 °C for at least 20 months. Any degradation from IFT to IFT-BA or from IFT-DKN to IFT-BA remains undetected, because of the common moiety method used.

Table 39 Storage stability of IFT, IFT-DKN and IFT-BA in sugarcane commodities

Commodity Storage time months

IFT % remaining mean, n=2–4 a


IFT-DKN % remaining mean, n=2–4 a


IFT-BA % remaining mean, n=2–4 a


Sugarcane billets 20 84 88 88 86 88 88 Sugarcane forage 20 79 93 85 95 82 93

a mean of two replicates for sugarcane, mean of four replicates for forage

Study 4

Storage stability was investigated in oranges, soya bean seeds, dry pinto beans, and sugarcane billets [Klasmeier, 2012, M-442915-01-1]. Homogenized untreated sample materials were individually spiked with IFT and IFT-DKN at a fortification level of 0.1 mg/kg. The samples were stored at –10 °C or lower for a period of 6 months. Samples were analysed in duplicate at various intervals using modification B of HPLC-MS-MS method IS-004-P10-01. Average concurrent recoveries for mixtures of IFT and IFT-DKN each at 0.1 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ).

Storage stability results and concurrent recoveries are shown in Table 40. The presented results for the stored samples are not corrected for the concurrent (freshly spiked) recoveries.

Additional results for this storage stability are available covering a storage period of 374-377 days (see Table 40) but there is no corresponding report available yet [Bayer, 2013].

Conclusion: After 3 months of storage at –10 °C, IFT partially converts to IFT-DKN in dry pinto beans while this conversion was observed at 6 months for oranges, soya bean seeds and sugarcane billets. Total residues (sum of IFT and IFT-DKN) and IFT-DKN alone remained stable for at least 1 year in each matrix tested. Storage stability of IFT-BA alone or further degradation of IFT or IFT-DKN to IFT-BA was not verified, but this is considered desirable.

No storage stability studies have been conducted on chickpea seeds, chickpea forage, chickpea straw, sweetcorn, maize forage, maize grains and maize fodder. Most of these commodities can be extrapolated from the present data: dry chickpea seeds vs dry pinto beans, chickpea forage vs sugarcane billets, sweetcorn vs sugarcane billets and maize forage vs sugarcane billets. Storage stability data on chickpea straw, maize grains and maize fodder cannot be extrapolated; these are desirable. Since maize forage, maize grains, maize fodder in the supervised residue trials have been stored for 593, 544 and 546 days, respectively, storage stability data for longer periods are desirable.

Table 40 Storage stability of IFT and IFT-DKN in various commodities

Commodity Storage time days


IFT concurrent recovery mean, n=2–3


IFT-DKN concurrent recovery, mean, n=2–3

Oranges 0 31 96 188 377 b

- 91 88 74 (IFT) + 21 (IFT-DKN) = 95 a 74 (IFT) + 19 (IFT-DKN) = 93 a

88 92 93 88 89

– 71 75 70 79

73 79 93 87 87

Pinto beans 0 36 94 184 374 b

– 71 58 (IFT) + 31 (IFT-DKN) = 90 a 49 (IFT) + 36 (IFT-DKN) = 85 a 48 (IFT) + 41 (IFT-DKN) = 89 a

95 97 88 71 70

– 80 75 77 75

81 92 90 73 74

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Soya bean seeds 0 35 94 183 374b

– 76 72 58 (IFT) + 24 (IFT-DKN) = 82 a 51 (IFT) + 31 (IFT-DKN) = 82 a

81 94 84 86 72

– 66 75 78 70

74 79 84 86 70

Sugarcane billets 0 29 95 185 375b

– 82 94 76 (IFT) + 9.0 (IFT-DKN) = 86 a 78 (IFT) + 12 (IFT-DKN) = 90 a

93 80 92 89 88

– 72 77 78 81

75 75 92 88 91

a Due to apparent partial conversion of IFT to IFT-DKN during storage, the recovery value reflects the sum of IFT and IFT-DKN (in isoxaflutole equivalents) b Additional results are available but there is no corresponding report available yet [Bayer, 2013].

Study 5

Storage stability was investigated for incurred residues in poppy seeds and poppy straw [Klempner, 2010, M-366215-01-1-01]. Poppy seeds and straw originated from the poppy metabolism study [Klempner, 2009, M1731775-1] where 100 g [phenyl-UL-14C]-Isoxaflutole per hectare was applied onto the bare soil (pre-emergence application). Samples from this study were stored at –18 °C or lower. Samples were extracted and analysed after 16.1 months of storage by HPLC-UV with radiodetection. The method used in the storage stability study was the same method used in the metabolism study.

Aliquots of 20 g of the homogenised samples were extracted three times with a mixture of ACN/water (8/2, v/v). Extracts were combined and filtered by suction. The TRR values of the poppy matrices were determined by summing up the radioactivity determined in the combined extracts and the radioactivity in the remaining solids after extraction. The extracts were subjected to a clean-up step using an SPE RP 18 cartridge (Varian Mega Bond Elut C18). The flow-through fraction (percolate) was collected and mixed with emulsifier and/or defoamer and evaporated to the aqueous remainder (= final extract). The final extracts were analysed by HPLC-UV with radiodetection. Storage stability was assessed IFT-DKN and IFT-BA, because parent IFT was not detected in poppy matrices.

Comparison of the metabolic profiles of the poppy seeds and poppy straw from the metabolism study (extraction after 16 days of storage for seeds and 12 weeks of storage for straw) and at 16.1 months showed that the distribution of components did not significantly change during this storage period. No significant transformation of compounds was found in this storage period. Residue results from both studies are shown in Table 41.

Conclusion: The reference time point for this storage stability study is the first analysis day in the metabolism study: 16 days for poppy seeds and 12 weeks for poppy straw. Since the reference point for poppy seeds is shortly after harvest, levels of IFT-DKN and IFT-BA in poppy seeds can be considered stable for a period of at least 16 months. No conclusion can be drawn for the storage stability in poppy straw, since residues have not been measured shortly after harvest. Consequently it is not clear, whether the analytes had already been degraded to a plateau level at the start of the analysis (i.e. 12 weeks after harvest). No storage stability information is available for IFT, but this is considered desirable.

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Table 41 Storage stability of incurred residues of IFT-DKN and IFT-BA in poppy seeds at -18 ºC

Matrix Storage period (days)

Origin TRR mg/kg eq

IFT-DKN (mg/kg eq)

IFT-DKN % remaining a

IFT-BA (mg/kg eq)

IFT-BA % remaining a

Poppy seeds 16 metabolism study 0.056 – – 0.037 – 482 storage stab study 0.056 – – 0.039 105 Poppy straw 84 metabolism study 0.725 0.026 – 0.643 – 482 storage stab study 0.721 0.027 104 0.630 98

a The concentrations found for isoxaflutole equivalent residue in the metabolism study were set to 100%.

Study 6

Homogenised samples of sorghum forage, sorghum fodder, sorghum grain, lettuce, radish leaves and radish roots were fortified with a mixture of 14C IFT, 14C-IFT-DKN and 14C-RPA203328 as total 2 mg/kg eq per sample [Hampton, 1996; M-192325-01-1]. Samples were stored at –20 ºC for 700 days. Duplicate samples were subsequently extracted with hexane/EtOAc (9:1 v/v), ACN, water (pH 5.5) and ACN/0.2 M HC1 (1:1 v/v). The pH for the water extraction was adjusted to pH 5.5, because at this pH hydrolysis of parent compound or conjugates did not occur. Radioactive residues in extracts were quantified by HPLC with radiodetection and co-chromatography of standards for IFT, IFT-DKN and IFT-BA.

Results are shown in Table 42. Extraction of day 0 spiked samples demonstrated a TRR of 99.3% present on average as 49% IFT, 33% IFT-DKN and 18% IFT-BA. After 700 days of storage the mean recovered TRR was 93.7% compared to 99.3% at day 0. The composition of the TRR changed. IFT was not sufficiently stable in storage, a decrease from 42.8–54.7% TRR (day 0) to 0.0–29.3% TRR after 700 days of storage was reported depending on matrix. Recovery for the intermediate metabolite IFT-DKN increased from 25.2–40.5% TRR (day 0) to 41.9–57.6% TRR (day 700) for most matrices, while a decrease of 34.2–40.5% TRR (day 0) to 26.4–31.4% TRR (day 700) was found in sorghum forage and sorghum grain. Also amounts of IFT-BA increased from 15.4–23.0% TRR at day 0 to 40.0–59.9% TRR after 700 days suggesting that degradation of Isoxaflutole to RPA202248 and subsequently from IFT-DKN to IFT-BA had occurred.

Conclusion: After 23 months of storage at –20 °C, isoxaflutole partially or completely converts to IFT-DKN and IFT-BA in sorghum forage, sorghum fodder, sorghum grain, lettuce, radish leaves and radish roots. Total residues (sum of IFT, IFT-DKN and IFT-BA) remained stable for at least 23 months. Storage stability of IFT-DKN alone or IFT-BA alone is masked by the isoxaflutole degradation. However, since the amount of IFT-BA increases with time, it indicates that IFT-DKN degrades further to IFT-BA. Storage stability studies with individual fortifications of IFT, IFT-DKN and IFT-BA are desirable.

Table 42 Storage stability of IFT, IFT-DKN and IFT-BA in various commodities


TRR IFT %TRR mean, n=2

IFT %remaining

IFT-DKN %TRR mean, n=2

IFT-BA %TRR mean, n=2

Sum a

%TRR

Sorghum forage 0 98.9 49.7 – 34.2 16.1 100.0 700 93.4 29.3 59 26.4 43.5 99.2 Lettuce 0 98.6 51.8 – 25.2 23.0 100.0 700 96.2 4.9 9.5 57.6 40.0 102.5 Radish leaf 0 99.1 54.7 – 26.4 19.0 100.1 700 94.1 0.0 0.0 48.7 51.0 99.7 Radish root 0 99.0 43.8 – 39.2 17.1 100.1 700 91.4 5.1 12 41.9 53.0 100.0 Sorghum grain 0 100.3 42.8 – 40.5 16.8 100.1 700 92.0 8.2 19 31.4 59.9 99.5 Sorghum fodder 0 99.9 50.0 – 34.7 15.4 100.1 700 95.0 12.9 26 46.1 40.9 99.9

Isoxaflutole

1231

a Sum of IFT, IFT-DKN and IFT-BA

Study 7

Storage stability was investigated in animal commodities purchased from a local food vender [Lowder, 1996, M-192281-01-2]. Milk samples were fortified with IFT, IFT-DKN, RPA 205834 and IFT-BA at 0.1 mg/kg each. Egg samples were fortified with IFT-DKN at 0.25 mg/kg. Bovine liver, kidney, muscle and fat samples were fortified with IFT, IFT-DKN, RPA 205834 and RPA 207048 at 0.25 mg/kg each. The storage samples were spiked individually with each analyte. The samples were stored frozen at –20 °C for up to 4 months. Samples were analysed at various intervals using HPLC-UV method RPAC #44882 for milk, HPLC-UV method RPAC #44933 for eggs and HPLC-MS-MS method RPAC #44996, version 2.0 for tissues. The analyses included a control sample, a freshly fortified control sample and duplicates of the storage samples.

For milk, the freshly fortified (i.e. concurrent recovery) samples were spiked with a mixture of IFT, IFT-DKN, RPA 205834, and IFT-BA at a level of 0.1 mg/kg for each analyte. For egg, the freshly fortified (i.e. concurrent recovery) samples were spiked with IFT-DKN at a level of 0.25 mg/kg. For liver, kidney, muscle, and fat tissues, two freshly fortified (i.e. concurrent recovery) samples were prepared: one with IFT alone, and one with a mixture of IFT-DKN, RPA 205834, and RPA 207048, each at 0.25 mg/kg. The tissue fortification procedure was necessary, because isoxaflutole is partially degraded to IFT-DKN during the tissue extraction procedure of HPLC-UV method RPAC #44996 and recovery of isoxaflutole in tissues can only be calculated as the sum of IFT and IFT-DKN. Levels in control samples were < 0.02 mg/kg (LOQ) in milk and < 0.05 mg/kg (LOQ) in eggs and tissues.

Storage stability results and concurrent recoveries are shown in Table 43 and Table 44. In these tables, the presented results for the stored samples are shown as not corrected and corrected (cr) by the concurrent (freshly spiked) recoveries. The power was off to the freezer for about 40 hours during the storage period. During this outage, temperatures rose to +5 °C. The outage would affect the "4 month" liver and muscle analyses.

Conclusion: The results for milk indicate that IFT-DKN, RPA 205834 and IFT-BA are stable for at least 127–128 days. Parent IFT is stable for a maximum period of 85 days, but degrades significantly thereafter (43% remaining at 127–128 days). The degradation of isoxaflutole on to IFT-DKN has not been verified. Storage stability of RPA 207048 has not been verified.

The results for eggs indicate that IFT-DKN is stable in the egg matrix for at least 129 days. Since parent IFT is immediately converted to IFT-DKN in the egg matrix, no fresh recovery of IFT is possible. Storage stability of metabolites RPA 205834 and RPA 207048 has not been verified.

The results for the tissues indicate that parent IFT and IFT-DKN are stable for at least 130 days in liver, 115 days in kidney, 113 days in fat, 131 days for IFT-DKN in muscle and a maximum period of 85 days for isoxaflutole in muscle. Metabolite RPA 205834 is stable for a period of 113–131 days in kidney, muscle, fat. Metabolite RPA 205834 is stable for a maximum storage period of 94 days in liver and degrades significantly thereafter (36% remaining after 130 days). Metabolite RPA 207048 is not stable in any animal commodity. RPA 207048 degrades instantaneously in kidney to a level of about 45% which is maintained from 13–115 days. RPA 207048 is stable for a maximum storage period of 28 days in muscle (70% remaining), a maximum period of 40 days in liver (74% remaining), and a maximum period of 84 days is fat (100% remaining) and thereafter remains stable at a level of about 50% of the original residue level for a period up to 85–113 days.

Isoxaflutole

1232

Table 43 Storage stability in milk and eggs

Matrix Storage time days






IFT-BA concurrent recovery, mean, n=2–3

RPA 205834 % remaining mean, n=2

RPA 205834 concurrent recovery, mean, n=2–3

Milk 0 15 29 56 85 127/128

– 66 (97 cr) 70 (81 cr) 71 (78 cr) 62 (81 cr) 43 (43 cr)

a 68 86 91 77 99

– 69 83 82 78 100

a 72 81 82 74 88

– 86 93 81 100 94

74 72 97 87 84 92

– 99 93 92 92 94

87 100 95 96 90 94

Eggs 0 15 29 56 88 129

– – – 98 96 85 91 92

95 99 99 95 101 95

– – – –

a Data not reported in the study report: the recoveries were too low to provide meaningful corrected results in the aged samples cr = corrected for concurrent recovery

Table 44 Storage stability for animal tissues

Matrix Storage time days

IFT % remaining mean, RSDr, n=2


IFT-DKN % remaining mean, RSDr, n=2

IFT-DKN concur rent recovery, mean, n=2–3

RPA 207048 % remaining mean, RSDr,n=2


RPA 205834 % remaining mean, RSDr,n=2


bovine liver

0 10 40 60 94 130 t

– 72 76 68 84 72

a 69 89 75 89 94

– 72 82 84 70 77

91 69 104 89 97 100

– 57 (90cr) 68 (74cr) 53 (61cr) 44 (57cr) 24 (29cr)

a 63 92 87 77 83

– 86 79 74 81 36

a 84 108 96 101 101

bovine kidney

0 13 27 62 84 115

– a b 88 86 88

72 a b 83 80 100

– 82 b 86 82 116

94 102 b 97 93 122

– 46 (48cr) b 45 (58cr) 44 (48cr) 42 (40cr)

88 95 b 77 91 104

– 96 b 82 78 102

a 96 b 93 95 116

bovine muscle

0 14 28 63 85 131 t

– a a 85 86 a

a a a 94 83 a

– 128 114 94 94 120

92 101 87 100 79 113

– 79 (98cr) 55 (70cr) 54 (62cr) 46 (62cr) 43 (45cr)

71 81 78 87 74 95

– a 86 90 74 86

82 a 90 100 97 99

bovine fat

0 13 28 62 84 113

– 82 81 84 82 88

a 97 79 80 85 84

– 66 100 88 94 98

70 120 89 82 95 120

– 85 (90cr) 74 (90cr) 56 (70cr) 79 (101cr) 51 (54cr)

69 94 82 80 78 94

– 88 88 94 87 94

73 89 89 92 90 110

a Data not reported in the study report: the recoveries were too low to provide meaningful corrected results in the aged samples b Data not reported in the study report: poor calibration curve for all analytes ( r2 < 0.99) due to shift in detector response during the run cr = corrected for concurrent method recovery t = The power was off to the freezer for about 40 hours during the storage period. During this outage, temperatures rose to +5 °C

Isoxaflutole

1233

USE PATTERN

The herbicide isoxaflutole belongs to the chemical class of isoxazoles and acts as a pigment photosynthesis inhibitor of 4-hydroxyphenyl-pyruvate-dioxygenase (4-HPPD). Isoxaflutole controls a wide spectrum of grasses and broadleaf weeds by bleaching emerging or emerged weeds following herbicide uptake via the root system.

Isoxaflutole is a registered herbicide in several countries and the original registered labels in the original language as well as their English translation were submitted for Australia, Canada, France, Hungary, Italy, Spain and the USA. Use patterns were submitted for sweetcorn, chickpeas, maize, sugarcane and poppy seed. An overview is presented in Table 45.

The Australian, Canadian and USA labels warn against crop damage:

� Soils with low organic carbon (OC) and low CEC have a reduced capacity to adsorb the herbicide in the soil, which may result in the herbicide leaching past the weed root zone into the crop root zone. Crop root uptake of isoxaflutole may result in phytotoxicity, which is evident as bleaching of leaves. To minimise the risk of sugarcane root uptake, isoxaflutole is not recommended for use on soil with a CEC below 4.5 meq/100 g, or an OC of 1.0% or less. Chickpeas planted in sandy or gravelly soils or soils low in clay or organic matter may result in crop damage. Maize or IFT-soya beans grown on loamy sands, sandy loams or sands and/or soils with less than 1.5–2.0% om or pH > 7.5 may result in crop damage. US labels have soil specific application rates: lowest dose rate for sand, loamy sand or sandy loam; medium dose rate for loam, silt loam, silt or sandy clay loam; highest dose rate for silty clay loam, clay loam, sandy clay, silty clay, or clay.

� Failure to thoroughly close and firm the seed furrow for chickpeas and maize may allow herbicide to directly contact the seed which may cause crop injury. Maize seeds need to be planted at least 4 cm deep.

� Isoxaflutole is activated by rainfall or irrigation, which is required to carry the herbicide into the root zone of the germinating weeds. Heavy rains after the application of isoxaflutole to chickpeas may cause crop damage, particularly in sandy or gravely soils.

Table 45 Registered pre-harvest uses of isoxaflutole

Crop Country Form g ai/kg g ai/L

Application PHI, days Method & Timing Rate

kg ai/ha Spray conc, kg ai/hL

No

chickpea Australia WG 750 ground based equipment only - just after planting (after furrow closure) to prior to emergence of the crop

0.075 < 0.15 1 seeds: NA a forage/fodder: at least 6 weeks after application

Maize: Field corn

Canada SC 240 ground based equipment only - pre-plant: up to 14 days prior to planting (soil surface treatment) - just after planting to prior to emergence of the crop - early post-emergence up to 3-leaf stage

0.079–0.106

< 0.070 1 grains: at least 110 days after any application forage/fodder: at least 60 days after application

Canada SC 480 ground based equipment only - pre-plant: 14–0 days prior to planting (soil surface treatment) - just after planting to

0.079–0.106

< 0.070 1 grains: at least 136 days after any application forage/fodder: at least 60 days after application

Isoxaflutole

1234




No

prior to emergence of the crop

Maize France SC 44 a post-sowing pre-emergence of crop - early post-emergence of crop at BBCH 00–13

0.099 0.033–0.050

1 NA

Maize Hungary SC 240 a 10–0 days before planting of the crop - after planting, pre-emergence of the crop - early post-emergence: up to 3 leaf stage of the crop

0.077–0.106

0.026–0.042

1 NA

Maize Italy SC 50 a, b

pre-emergence of crop - early post-emergence up to 3 leaf stage of the crop

0.075–0.100

0.019–0.050

1 NA

Maize Spain WG 750 normal spray - pre-emergence of crop

0.052–0.10

0.010–0.051

1 NA

Maize: Field corn

USA WG 750 e

ground based equipment for broadcast or banded applications - pre-plant: 30–0 days before planting maize (surface applied or incorporated in the soil at < 5 cm deep) - pre-emergence: during planting (after furrow closure) or before crop emerges

0.053–0.16

< 0.17 1 or 2 f

NA

USA SL 479 (40.5% w/w) e

ground based equipment for broadcast or banded applications - pre-plant: 30–0 days before planting (surface applied or incorporated in the soil at < 5 cm deep) - pre-emergence: during planting (after furrow closure) or before crop emerges

0.053–0.105

< 0.11 1 or 2 f

NA

USA SC 225 (19.0% w/w) a, b, e

ground based equipment for broadcast or banded applications - pre-plant: 30–0 days before planting (surface applied or incorporated in the soil at < 5 cm deep) - pre-emergence: during planting (after furrow closure) or before crop emerges - early post-emergence up to 2 leaf-collar growth stage of maize (V2,

0.055– 0.092

< 0.099 1 grains: NA forage: at least 45 days after early post-emergence treatment

Isoxaflutole

1235




No

where the first leaf has a rounded tip)

USA SC 240 (20.0% w/w a, e

ground based equipment for broadcast or banded applications - pre-plant: 30–0 days before planting (surface applied or incorporated in the soil at < 5 cm deep) - pre-emergence: during planting (after furrow closure) or before crop emerges - early post-emergence up to 2 leaf-collar growth stage of maize (V2, where the first leaf has a rounded tip)

0.053– 0.105

< 0.11 1 or 2 f

grains: NA forage: at least 45 days after early post-emergence treatment

sweetcorn France SC 44 a post sowing pre-emergence of crop at BBCH 00–08

0.099 0.033–0.050

1 NA

poppy seed

Hungary SC 240 a pre-emergence: within 2–3 days after planting of the crop

0.077–0.106

0.026–0.042

1 NA

Spain WG 750 normal spray - pre-emergence of crop

0.045– 0.060

0.009– 0.030

1 NA

sugar cane

Australia WG 750 c ground based equipment only d -Pre- to early post-emergence of plant cane (up to 3–4 leaf crop stage) applied as broadcast or band spray over the top of the plant cane-After harvest of ratoon cane (up to 2 leaf crop stage) applied as broadcast or band spray - Prior to the “out-of-hand” stage of plant and ratoon cane applied as directed inter-row spray to the soil surface (sugarcane > 0.75 m height)

0.150 < 0.060 1 canes: at least 19 weeks after application forage: do not graze animals on treated crops

SC (suspension concentrate), WG (water dispersible granule), SL (soluble concentrate) NA: PHI not applicable, because of the restriction in timing of the application a This product also contains the safener cyprosulfamide b This product also contains the herbicide thiencarbazone-methyl. c Do not apply with wetting agents, crop oils or adjuvants d Do not apply to soils with CEC below 4.5 meq/100 g or < 1.0% organic carbon. e Crop oil concentrate or methylated seed oil is recommended when weeds are present at the time of treatment. f Either one application with 100% of the recommended rate can be made at the timing period indicated or two split applications can be made with 60% of the recommended broadcast rate applied 15 to 30 days prior to planting (pre-plant surface applied) and the remaining 40% applied at planting.

Isoxaflutole

1236

RESIDUES RESULTING FROM SUPERVISED TRIALS ON CROPS

The Meeting received information on supervised residue trials of isoxaflutole for the following crops:

Group Table Commodity

Fruiting vegetables other than cucurbits 46 Sweet corn

Pulses 47 Chickpea seeds

48 Glyphosate/HPPD tolerant soya beans seeds

Cereals 49 Maize grains

Grasses for sugar or syrup production 50 Sugarcane billets

Oilseeds 51 Poppy seeds

Legume animal feeds 52 Chickpea forage

53 Chickpea straw

Straw, fodder and forage of cereal grains and grasses 54 Maize forage

55 Maize fodder

Miscellaneous forage and fodder crops 56 sugarcane forage

Application rates and spray concentrations have been rounded to three figures; residues have been rounded to two figures. Residue data are recorded unadjusted for percentage recoveries or for residue values in control samples unless otherwise stated. Unquantifiable residues are shown as below the reported LOQ (e.g. < 0.01 mg/kg). Where multiple samples were taken from a single plot or where multiple analyses were conducted on a single sample, the average value is reported. Where results from separate plots with distinguishing characteristics such as different formulations, crop varieties or treatment schedules were reported, results are listed separately for each plot. Residues from the trials conducted according to critical GAP have been used for the estimation of maximum residue levels, STMR and HR values. Those results are underlined.

The residues presented in the tables are given as individual compound (and expressed as such) or as total residue. The total residue represents the sum of parent + IFT-DKN, expressed as isoxaflutole, unless stated otherwise. Since the relative molecular weight of IFT-DKN is the same as the relative molecular weight of isoxaflutole, no conversion for molecular weight is necessary. Where residues of were < 0.01 mg/kg, these were calculated as being at 0.01 mg/kg (expressed as metabolite).

Sweet corn

Supervised trials on maize were conducted in Spain, Italy, Greece, Portugal, Germany, France, Netherlands and the United Kingdom in 2005 and 2006 [Zimmer and Wieland, 2007, M-282674-01-1, report RA-2587/05; Wolters and Erler, 2007b, M-284423-01-1, report RA-2510/06; Wolters, 2007b, M-285014-01-1, report RA-2615/06; Zimmer, 2006, M-281611-01-1, report RA-2588/05; Wolters and Erler, 2007a, M-284416-01-1, report RA-2511/06; Wolters, 2007a, M-285005-01-1, report RA-2616/06]. Small plots of maize (50–1700 m2) were treated as indicated in Table 46 using knapsack, tractor/ATV mounted or wheelbarrow sprayers with spray volumes ranging from 90–300 L/ha

Four treatment scenarios were tested in the plots:

� a single application of isoxaflutole in combination with the safener cyprosulfamide at BBCH 12–14,

� a single application of isoxaflutole in combination with thiencarbazone-methyl and the safener cyprosulfamide at BBCH 13–14.

Isoxaflutole

1237

� a single bare ground application of isoxaflutole in combination with the safener cyprosulfamide (pre-emergence of the crop) followed by a second application with thiencarbazone-methyl in combination with the safener cyprosulfamide at BBCH 18.

Single field samples of ears without husks were taken as “baby corn” (BBCH 61), as “sweet corn” (milk ripeness, BBCH 79) or as “silage stage corn” (BBCH 85). All sample sizes were at least 2 kg and at least 12 items (in accordance with FAO manual 2009), except for some baby corn samples (0.6–1.2 kg) as indicated in Table 46. All samples were stored frozen with a maximum storage period of 244 days (8 months) at -18 ºC.

Isoxaflutole and its metabolites IFT-DKN and IFT-BA were determined using HPLC-MS-MS method 00985/M001. This method is considered valid in the range 0.01–1.0 mg/kg for sweet corn. Average concurrent recoveries at 0.01–1.0 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (LOQ) for each analyte. Average concentrations for single field samples are shown in Table 46.

Note: Storage stability studies on commodities with high water content showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 20 months. Storage stability studies on commodities with high water content showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 12 months but converts to IFT-BA within 23 months of storage. Since sweet corn has been stored for less than 12 months residues are considered reliable.

Note: All trials listed in Table 46 on sweet corn have been conducted with the safener cyprosulfamide. The manufacturer submitted summaries of six European trials (three from southern Europe and three from northern Europe) for sweet corn for the use of isoxaflutole in pre-emergence at 0.10 or 0.30 kg ai/ha without the safener cyprosulfamide, where it is shown that the presence of the safener cyprosulfamide does not affect the residue levels of IFT-DKN and IFT-BA. In these additional trials, the residue data were measured using a common-moiety method (sum of isoxaflutole, IFT-DKN and IFT-BA, expressed as isoxaflutole). In all trials, residue levels in the ear/cob harvested at DAT 76–158 were found to be < 0.013 mg/kg eq [Bayer, 2013]. These trials were not further investigated, since they have been conducted with a common moiety method and they confirm the residues found in the studies in Table 46.

Table 46 Isoxaflutole related residues after post-emergent treatment in sweet corn (ears without husks)

SWEET CORN Location, year, (variety)

FL no kg ai/ha

kg ai/hL

GS1 GS2 DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total residues mg/kg eq

Study Trial

Germany Monheim (Nordrhein-Westfalen) 2005, (Romario) Soil: SaL

SC

a 1 0.10 0.034 13 79

85 78 111

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05; R 2005 0623/6

Germany Monheim (Nordrhein-Westfalen) 2006, (Romario), Soil: SaSi

SC

b 1 0.099 0.033 13 61

79 55 d 77

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2510/06 R 2006 0795/4

Germany Euskirchen - Kessenich (Nordrhein-Westfalen), 2005 (Egrin FAO 220), Soil: SiC

SC

a 1 0.10 0.034 13 79

85 90 112

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05 R 2005 0961/8

Germany Leverkusen (Nordrhein-Westfalen),

SC

c 1 c 0.10 0.034 05 61

79 71 d 91

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006

Isoxaflutole

1238


FL no kg ai/ha

kg ai/hL


IFT-DKN mg/kg

IFT-BA mg/kg


Study Trial

2006 (Bunguy); Soil: SaL

0801/2

Germany Werl (NordrheinWestfalen), 2006, (Delitop); Soil: SiL

SC

c 1 c 0.10 0.034 06 61

79 80 d 116

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0802/0

United Kingdom Cambridge (Cambridgeshire), 2005, (RK210), Soil: SaL

SC

a 1 0.10 0.034 13 79

85 103 124

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05 R 2005 0959/6

United Kingdom Cambridge (Cambridgeshire) 2006, (Nexxos); Soil: SaL

SC

b 1 0.099 0.033 14 61

79 51 d 93

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2510/06 R 2006 0796/2

United Kingdom, EYE (Suffolk) 2006, (Algans); Soil: SiL

SC

c 1 c 0.10 0.034 07 61

79 84 d

101 < 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0800/4

Netherlands Zwaagdijk-Oost (Noord-Holland) 2005, (Rosalie), Soil: C

SC

a 1 0.10 0.034 13 79

85 116 128

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05 R 2005 0962/6

N-France Saint Symphorien d'Ancelles (Bourgogne) 2005, (Surtep), Soil: S

SC

a 1 0.10 0.034 14 79

85 71 83

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05 R 2005 0958/8

N-France Chaussy (Ile-de-France) 2006, (Moncada), Soil: CSa

SC

b 1 0.099 0.033 13 61

79 62 96

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2510/06 R 2006 0073/9

- SC

c 1 c 0.10 0.034 01 61

79 86 120

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0627/3

N-France Chambourg sur indre (Centre) 2006, (Anasta); Soil: CSi

SC

c 1 c 0.10 0.034 00 61

79 89 d 112

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0799/7

S-France Savenes (Midi-Pyrenees), 2005, (PR33A46); Soil: CL

SC

a

1 0.10 0.034 13 79 85

83 106

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0624/4

S-France Vouille (Poitou-Charentes)

SC

b 1 0.099 0.033 13 61

79 74 d 98

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2511/06 R 2006

Isoxaflutole

1239


FL no kg ai/ha

kg ai/hL


IFT-DKN mg/kg

IFT-BA mg/kg


Study Trial

2006, (dkc 4845) Soil: SiC

0074/7

S-France Beauzelle (Midi-Pyrenees) 2006, (Ferry) Soil: SaSi

SC

c 1 c 0.10 0.034 06 61

79 79 d 105

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0628/1

S-France Laudun (Languedoc-Roussillon) 2006, (Cecilia) Soil: CL

SC

c 1 c 0.10 0.034 01 61

79 64 d 91

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0803/9

Spain Vila-sacra (Cataluña) 2005, (DKc6575); Soil: SaL

SC

a 1 0.10 0.034 13 79

85 84 98

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0963/4

Spain Vila-sacra (Cataluña), 2006, (PR33P67) Soil: SaL

SC

b 1 0.099 0.033 14 61

79 56 d 67

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2511/06 R 2006 0798/9

SC

c 1 c

0.10 0.034 07 61 79

78 d 89

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0804/7

Spain Alginet (Comunidad Valenciana) 2006, (Constanza) Soil: SiC

SC

c 1 c

0.10

0.034 00 61 79

65 78

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0806/3

Italy San Carlo (FE), Emilia-Romagna 2005, (DK 440); Soil: SaC

SC

a 1 0.10 0.034 13 79

85 79 93

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0964/2

Italy San Carlo (FE), Emilia-Romagna 2006, (PR34N43) Soil: SaC

SC

b 1 0.099 0.033 13 61

79 53 d 75

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2511/06 R 2006 0797/0

SC

c 1 c

0.10 0.034 01 61 79

78 d 100

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0805/5

Greece Chalastra (Macedonia) 2005, (Decalp 743); Soil: L

SC

a 1 0.10 0.034 13 79

85 77 105

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0965/0

Portugal Golegã (Ribatejo e Oeste) 2005, (PRN 43); Soil: SiL

SC

a 1 0.10 0.034 13 79

85 80 100

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0966/9

Isoxaflutole

1240

GS: BBCH grows stage: GS1 - at application; GS2 - at harvest Soil: C = Clay, L= loam, Si = silt; Sa= sand; CL = clay loam; CSa = clay sand, CSi= clay silt; LSa = loamy sand; SiC = silty clay; SaCL=sandy clay loam; SiCL = silty clay loam, SiL = silty loam; SaC = sandy clay; SaL = sandy loam, SaSi = sandy silt; Total = sum of IFT and IFT-DKN, expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. a SC formulation contained 20.4% w/w isoxaflutole and 20.3% w/w cyprosulfamide (safener); or 240 g/L each b SC formulation contained 225 g/L isoxaflutole, 150 g/L cyprosulfamide (safener) and 90 g/L thiencarbazone-methyl c A first spray application was performed with an SC formulation (containing 240 g/L isoxaflutole and 240 g/L cyprosulfamide (safener)) at the pre-emergence stage followed by a second application with an SC formulation (containing 225 g/L thiencarbazone-methyl and 225 g/L cyprosulfamide (safener)) at growth stage BBCH 18. Days after last treatment (DAT) are related to the isoxaflutole treatment (i.e. days after the first treatment). d Sample sizes were less than the required minimum of 2 kg (in accordance with FAO manual 2009): 0.6–1.1 kg at BBCH 61 (baby corn) in reports RA-2510/06, RA-2615/06, RA-2511/06. Samples are considered not representative and trials cannot be selected for MRL derivation, if according to cGAP.

Pulses

Supervised trials were conducted on chickpeas and glyphosate/HPPD tolerant soya beans.

Chickpea seeds (dry harvested)

Supervised trials on chickpeas were conducted in Australia during the 1999 and 2011 season [Lynch and Keats, 1999a, M-432364-01-1, report AK99054; Lynch and Keats, 1999b, M-432374-01-1, report AK99056; Lynch and Keats, 2000a, M-432434-01-1, report AQ00001; Lynch and Keats, 2000b, M-432441-01-1, report AQ00002; Lynch and Keats, 2000c, M-432444-01-1, report AQ00003; Ellis, 2012, M-425905-02-1, report BCS-0370]. Small plots of chickpeas (20–135 m2) were treated as indicated in Table 47 using a hand held boom sprayer or a small plot sprayer with spray volumes ranging from 50–230 L/ha.

In 1999, different application scenarios were tested on parallel plots. Plots were treated once at different application rates and/or growth stages (before emergence of the crop or at 3 nodes stage).

In 2011, four application scenarios were tested on parallel plots. Isoxaflutole was applied at rates of 0.075 kg ai/ha and 0.150 kg ai/ha to bare soil either as a single fallow application approximately four month before sowing the chickpea crops, or in conjunction with a second application at planting.

Replicate field samples of dry pea seeds were taken at maturity (marketable specimens). In report AK99054, chickpea plots were mechanically harvested and seeds were randomly selected from a harvester bin. In report AK99056, BCS-370 and AQ reports dry pea seeds were sampled by hand, cutting random samples of chickpea pods from at least 10 separate plants from all over the plot. Sample sizes were at least 1 kg in report AQ00003 and AK99054. Sample sizes from all other reports were below the required minimum of 1 kg (in accordance with FAO manual 2009): 0.1 kg in report AK99056, 0.25 kg in AQ00001 and AQ00002, 0.5 kg in BCS-370. Samples were stored frozen with a maximum storage period of 2–21 days at –20 ºC (AK99054 and AQ reports), 11 months at -20 ºC (AK99056) or 21 days at -8 ºC (BCS-0370).

In the trials from 1999, samples were analysed for total residues using GC-MS-MS modification B of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and total residues are expressed as isoxaflutole equivalents. This method is considered valid in the range 0.01–0.1 mg/kg for dry chickpea seeds. Average concurrent recoveries at 0.01 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3LOQ). Average concentrations for duplicate field samples are shown in Table 47.

In the trials from 2011, samples were analysed for IFT, IFT-DKN and IFT-BA using modification A of HPLC-MS-MS method ATM-0054.01. This method is considered valid in the range 0.01–0.1 mg/kg in dry chickpea seeds. Average concurrent recoveries at 0.01–1.0 mg/kg were

Isoxaflutole

1241

within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 47.

Note: In the AK and AQ reports, some data (application rate and date of application) were found to be inconsistent between the summary and appendices. The data coming from the field presented in the “residue sample submission form” were considered as being the valid ones.

Note: Storage stability studies on commodities with high protein content showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 11 months. Storage stability studies on commodities with high protein content showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 12 months but converts to IFT-BA within 23 months of storage. Since chickpeas have been stored for a maximum of 11 months in the trials where a common moiety method was used and chickpeas have been stored for less than 3 months in trials where individual compounds have been measured, all results are considered reliable.

Table 47 Isoxaflutole related residues after pre- or post-emergent treatment of chickpeas (dry harvested seeds)

CHICK PEA Location, year, (variety)

Form No & interval d

kg ai/ha kg ai/hL timing DAT IFT, mg/kg

IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg eq

report, trial

Australia Pittsworth QLD, 1999 (Norwin) Soil: black earth

WG 1 0.075 0.075 Post planting, pre-emergent

185 NA NA NA < 0.01 a AK99054 99NST26-A


185 NA NA NA < 0.01 a

AK99054 99NST26-B

Australia Biniguy NSW, 1999, (Amethyst) Soil: brown clay loam

WG 1 0.075 0.100 3 days post sowing

155 NA NA NA < 0.01 a AK99056 99NST01-A c


155 NA NA NA < 0.01 a AK99056 99NST01-B c

Australia Horsham VIC, 1999, (Sona) Soil: Wimmera gray clay

WG 1 0.075 0.100 Post sow/ Pre-emergent

184 NA NA NA < 0.01 a AQ00001 99h011-A

c


184 NA NA NA < 0.01 a AQ00001 99h011-B

c WG

d 1 0.056 0.075 Early

Post-emergent (3 nodes)

147 NA NA NA < 0.01 a AQ00002 99h015-A

c

WG d

1 0.075 0.100 Early Post-emergent (3 nodes)

147 NA NA NA < 0.01 a AQ00002 99h015-B

c

WG d

1 0.094 0.13

Early Post-emergent (3 nodes)

147 NA NA NA < 0.01 a AQ00002 99h015-C

c

Isoxaflutole

1242




IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg eq

report, trial

WG e 1 0.056 0.075 Early Post-emergent (3 nodes)

147 NA NA NA < 0.01 a AQ00002 99h015-D

c


147 NA NA NA < 0.01 a AQ00002 99h015-E

c


147 NA NA NA < 0.01 a AQ00002 99h015-F

c

Australia Bruce Rock WA, 1999, (Tyson) Soil: brown loam

WG d 1 0.075 0.15 Post sow/Pre-emergent

188 NA NA NA < 0.01 a AQ00003 99h010-


188 NA NA NA < 0.01 a AQ00003 99h010-B


188 NA NA NA < 0.01 a AQ00003 99h010-C

WG e 1 0.075 0.15 Post sow/Pre-emergent

188 NA NA NA < 0.01 a AQ00003 99h010-D


188 NA NA NA < 0.01 a AQ00003 99h010-E


188 NA NA NA < 0.01 a AQ00003 99h010-F

Australia Northam WA, 2011, (Desi) Soil: sandy loam

WG 1 0.075 0.033

bare soil 121 days before sowing

295 < 0.01 < 0.01 < 0.01 < 0.02 b BCS-0370, C601 c

WG 1 0.15 0.066 bare soil 121 days before sowing

295 < 0.01 < 0.01 < 0.01 < 0.02 b c

WG 2 (121 days)

0.075 0.075

0.0330.075

1st bare soil, 2nd at sowing

174 < 0.01 < 0.01 < 0.01 < 0.02 b c

WG 2 (121 days)

0.15 0.15

0.0660.150


174 < 0.01 < 0.01 < 0.01 < 0.02 b c

Australia Warwick QLD, 2011, (Hattrick) Soil: black clay loam


284 < 0.01 < 0.01 < 0.01 < 0.02 b BCS-0370, C602 c


284 < 0.01 < 0.01 < 0.01 < 0.02 b c

WG 2 (127

0.075 0.075

0.0550.057

1st bare soil, 2nd at

157 < 0.01 < 0.01 < 0.01 < 0.02 b c

Isoxaflutole

1243




IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg eq

report, trial

days) sowing WG 2

(127 days)

0.15 0.15

0.11-0.11


157 < 0.01 < 0.01 < 0.01 < 0.02 b c

Australia Shepparton East, VIC, 2011, (Kabul) Soil: clay loam


324 < 0.01 < 0.01 < 0.01 < 0.02 b BCS-0370, C603 c


324 < 0.01 < 0.01 < 0.01 < 0.02 c c

WG 2 (107 days)

0.0750.072

0.0750.075


217 < 0.01 < 0.01 < 0.01 < 0.02 b c

WG 2 (107 days)

0.15 0.15

0.150 1st bare soil, 2nd at sowing

217 < 0.01 < 0.01 < 0.01 < 0.02 b c

a Total residue obtained by common moiety method, whereby residues are converted into RPA 204497 and expressed as isoxaflutole equivalent (residue includes IFT, IFT-DKN and IFT-BA) b Total residue calculated by summing up residues of IFT and IFT-DKN and expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. c Sample sizes were below the required minimum of 1 kg (in accordance with FAO manual 2009): 0.1 kg in report AK99056, 0.25 kg in AQ00001 and AQ00002, 0.5 kg in BCS–370. However, two samples were taken from each trial plot (each from at least 10 plants in the AK and AQ reports) and the results are the mean of both samples. Therefore results can be considered representative and trials can be selected for MRL derivation, if according to cGAP. d Added adjuvant: 0.25% X-77 e Added adjuvant: Uptake oil

Glyphosate/HPPD tolerant soya beans

Supervised trials on glyphosate/HPPD tolerant soya beans were conducted in the USA and Canada during the 2009 season [Dallstream and Fisher, 2010, M-368661-01-1, study RAISP006; Beedle and Dallstream, 2010, M-368669-01-1, study RAISP010]. Small plots of soya beans (500–1800 ft2 or 46–167 m2) were treated as indicated in Table 48 using backpack or tractor mounted sprayers with spray volumes ranging from 9–26 GPA or 84–243 L/ha.

In report RAISP006 three different application scenarios were tested in parallel plots: a) single pre-plant or pre-emergence soil application, b) single foliar application from BBCH 13 (trifoliate leaf on the 3rd node unfolded) to BBCH 15 (trifoliate leaf on the 5th node unfolded) or c) single foliar application from BBCH 51 (first flower buds visible) to BBCH 66 (about 60% of flowers open). The pre-emergent applications occurred within 3 days following planting.

In report RAISP010 two different application scenarios were tested on parallel plots:

� In the first plot a single pre-emergent application of glyphosate-isoxaflutole spray mix was made, followed by two additional foliar applications of glyphosate.

� For the second plot, a single pre-emergent application of glyphosate was made, followed by a single foliar application of glyphosate-isoxaflutole spray mix between growth stages BBCH 51 (first flower buds visible) and BBCH 62 (about 20% of flowers open), and an additional foliar application of glyphosate 8–11 days later.

The pre-emergent applications occurred within 3 days following planting.

Isoxaflutole

1244

Replicate field samples of soya bean seed were collected at earliest commercial harvest (BBCH 89). Seed samples were a composite from at least 12 areas of the plot, avoiding the edges and ends of the plot. Samples sizes were 1 kg (in accordance with FAO manual 2009), except for trial IS007-09DA (0.20–0.44 kg). All samples were stored frozen with a maximum storage period of 188 days at 0 ºC (or lower in report RAISP006) or 218 days at –20 ºC (or lower in report RAISP010).

Samples were analysed for IFT and IFT-DKN using HPCL-MS-MS method IS-004-P10-01. The method is considered valid in the range 0.01–0.1 mg/kg in dry soya bean seed. Average concurrent recoveries at 0.01 and 0.1 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ) for each analyte. Average concentrations for duplicate field samples are shown in Table 48.

Note: Storage stability studies on various commodities showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 11–20 months. Storage stability studies on commodities with high oil content showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 16 months but converts to IFT-BA within 23 months of storage. Since dry soya beans have been stored for longer than 6 months, part of the IFT may have degraded to IFT-DKN. Since dry soya beans have been stored for less than 16 months, levels of IFT-DKN are not further degraded to IFT-BA and can be considered reliable. As the residue definition includes both IFT and IFT-DKN, residues can be taken for MRL derivation, if according to cGAP.

Note: Isoxaflutole inhibits the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) while glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The glyphosate/HPPD tolerant soya bean line (FG72) was produced by direct gene transfer of the transgenic insert to conventional soya bean cells. FG72 soya bean expresses the hppdPfW336 gene from Pseudomonas fluorescens and the 2mepsps gene derived from maize. These genes confer tolerance to the herbicide isoxaflutole and glyphosate-containing herbicides, respectively [COGEM advice CGM/120104-02].

Note: Crops can be made tolerant against herbicides in two different ways:

� Some tolerant crops are able to detoxify the herbicide via a specific chemical modification of the pesticide which makes it unable to react with its molecular target. The detoxification is usually mediated by an enzyme which is very specific for the herbicide under consideration. Interaction with other pesticides cannot be completely excluded but normally will not occur at high level. Thus a significant impact on the metabolism and residue behaviour of other pesticides is quite unlikely.

� Other crops are made tolerant to herbicides via a modification of the target enzyme which makes the modified enzyme insensitive against the herbicide. The FG72 soya bean variety contains a gene coding for the modified enzyme HPPDW336 which differs only by a single amino acid from the natural HPPD enzyme. While HPPDW336 has the same biological activity as HPPD it is not deactivated by isoxaflutole. The FG72 soya bean variety also contains a gene for the modified enzyme 2mEPSPS which has a biological activity comparable to that of the natural EPSPS but is not deactivated by glyphosate.

Metabolism studies and field trials all have been conducted with the glyphosate/HPPD tolerant soya bean line (FG72). No trials have been conducted with a soya bean variety where only the HPPD tolerant gene is present. Since in the FG72 soya bean variety the tolerance against glyphosate and isoxaflutole is not based on detoxification, the tolerance to glyphosate is not expected to modify the nature and levels of isoxaflutole-derived residues in treated soya beans. Similarly the tolerance to isoxaflutole is not expected to modify the nature and levels of glyphosate residues.

Isoxaflutole

1245

Table 48 Isoxaflutole related residues after pre-emergent or foliar treatment on soya beans (dry harvested seeds)

SOYA BEAN Location, year, (variety), soil type

Form No kg ai/ha kg ai/hL GS appl

DAT IFT, mg/kg

IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg eq

% DM

Report, trial, (remarks)

Canada Rockwood ON, 2009, (FG 72) Soil: L

SC b 1 0.103 0.0786 00 151 < 0.01 < 0.01 NA < 0.02 80 RAISP006 IS008-09HA

1 0.130 0.0663 15 120 < 0.01 0.011 NA 0.021 81 1 0.106 0.0675 51 99 < 0.01 < 0.01 NA < 0.02 79 SC a, b 1 0.106 0.0791 00 90 < 0.01 < 0.01 NA < 0.02 80 RAISP01

0 IS035-09HA

1 0.106 0.0803 51 90 < 0.01 < 0.01 NA < 0.02 83 USA, Chula, GA, 2009 (FG 72), soil LSa

SC b 1 0.103 0.0551 00 144 < 0.01 < 0.01 NA < 0.02 91 RAISP006, IS002-09HA

1 0.105 0.0461 15 120 < 0.01 < 0.01 NA < 0.02 89 1 0.104 0.0477 66 98 < 0.01 0.021 NA 0.031 89 USA, Shorter, AL, 2009, (FG 72) soil LSa

SC b 1 0.103 0.0673 01 120 < 0.01 < 0.01 NA < 0.02 85 RAISP006, IS003-09HA

1 0.103 0.0682 15 94 < 0.01 < 0.01 NA < 0.02 85 1 0.103 0.0673 62 82 < 0.01 0.016 NA 0.026 81 USA Shorter AL, 2009, (FG 72) Soil SaL

SC a, b 1 0.103 0.0673 01 72 < 0.01 < 0.01 NA < 0.02 85 RAISP010 IS032-09HA

1 0.103 0.0673 62 72 < 0.01 0.027 NA 0.037 85 USA, Cheneyville, LA,2009, (FG 72) soil SiL

SC b 1 0.107 0.0787 00 128 < 0.01 < 0.01 NA < 0.02 77 RAISP006 IS004-09DA

1 0.105 0.0673 13 106 < 0.01 < 0.01 NA < 0.02 79 1 0.106 0.0711 59 87

89 91 93 95

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

NA NA NA NA NA

< 0.02 < 0.02 < 0.02 < 0.02 < 0.02

88 73 78 85 88

USA, Greenville, MS, 2009, (FG 72) soil CL

SC b 1 0.104 0.0754 00 132 < 0.01 < 0.01 NA < 0.02 89 RAISP006 IS005-09HA

1 0.105 0.0789 15 110 < 0.01 < 0.01 NA < 0.02 88 1 0.105 0.0820 61 126 < 0.01 < 0.01 NA < 0.02 89 USA Proctor, AR, 2009, (FG 72) Soil CL

SC b 1 0.104 0.0717 00 111 < 0.01 < 0.01 NA < 0.02 85 RAISP006 IS006-09HA

Isoxaflutole

1246



DAT IFT, mg/kg

IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg eq

% DM


1 0.104 0.0717 15 87 < 0.01 < 0.01 NA < 0.02 70 1 0.104 0.0717 60 72 < 0.01 < 0.01 NA < 0.02 87 SC a, b 1 0.104 0.0717 00 62 < 0.01 < 0.01 NA < 0.02 89 RAISP01

0 IS033-09HA

1 0.104 0.0722 60 62 < 0.01 < 0.01 NA < 0.02 87 USA, Jefferson, IA, 2009, (FG 72) Soil SaL

SC b 1 0.107 0.13 00 130 < 0.01 < 0.01 NA < 0.02 82 RAISP006 IS007-09DA c

1 0.104 0.0765 13 94 < 0.01 < 0.01 NA < 0.02 89 c 1 0.105 0.0621 51 77

79 81 83 85

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 0.014 0.010 0.011

NA NA NA NA NA

< 0.02 < 0.02 0.024 0.020 0.021

84 82 82 87 85

c

USA Richland IA, 2009, (FG 72) Soil SiCL

SC b 1 0.105 0.0714 00 138 < 0.01 < 0.01 NA < 0.02 81 RAISP006 IS015-09HA

1 0.098 0.054 13 115 < 0.01 < 0.01 NA < 0.02 80 1 0.103 0.0589 60 95 < 0.01 < 0.01 NA < 0.02 78 USA Farlin IA, 2009, (FG 72) Soil CL


1 0.104 0.13 60 77 < 0.01 0.015 NA 0.025 89 USA Marysville OH, 2009, (FG 72) Soil CL

SC b 1 0.103 0.0610 00 139 < 0.01 < 0.01 NA < 0.02 91 RAISP006 IS009-09HA


0 IS036-09HA

1 0.105 0.0644 60 78 < 0.01 0.017 NA 0.027 91 USA Dudley MO, 2009, (FG 72) Soil SiL

SC b 1 0.106 0.0564 05 123 < 0.01 < 0.01 NA < 0.02 85 RAISP006 IS010-09HA

1 0.106 0.0567 15 98 < 0.01 < 0.01 NA < 0.02 86 1 0.105 0.0565 60 88 < 0.01 0.012 NA 0.022 84 USA Clarence MO, 2009, (FG 72) Soil SiCL

SC b 1 0.106 0.0573 00 129 < 0.01 < 0.01 NA < 0.02 87 RAISP006 IS011-09HA


Isoxaflutole

1247



DAT IFT, mg/kg

IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg eq

% DM


0 IS038-09HA

1 0.107 0.0549 60 80 < 0.01 < 0.01 NA < 0.02 90 USA Campbell MN, 2009, (FG 72) Soil SaCL

SC b 1 0.105 0.0561 00 130 < 0.01 < 0.01 NA < 0.02 82 RAISP006 IS012-09HA

1 0.104 0.0556 15 95 < 0.01 < 0.01 NA < 0.02 82 1 0.105 0.0561 60 88 < 0.01 < 0.01 NA < 0.02 82 SC a b 1 0.105 0.0561 00 79 < 0.01 < 0.01 NA < 0.02 82 RAISP01

0 IS039-09HA

1 0.105 0.0561 60 79 < 0.01 < 0.01 NA < 0.02 86 USA Geneva MN, 2009, (FG 72) Soil SaCL

SC b 1 0.106 0.0688 00 151 < 0.01 < 0.01 NA < 0.02 78 RAISP006 IS019-09HA

1 0.105 0.0618 15 116 < 0.01 < 0.01 NA < 0.02 73 1 0.105 0.0636 60 92 < 0.01 < 0.01 NA < 0.02 82 USA York NE, 2009, (FG 72) Soil SiL

SC b 1 0.107 0.0569 00 139 < 0.01 < 0.01 NA < 0.02 90 RAISP006 IS013-09HA


0 IS040-09HA

1 0.105 0.0555 60 89 < 0.01 < 0.01 NA < 0.02 89 USA Springfield NE, 2009, (FG 72) Soil SiL

SC b 1 0.107 0.0829 00 140 < 0.01 < 0.01 NA < 0.02 89 RAISP006 IS020-09HA

1 0.103 0.0798 15 112 < 0.01 < 0.01 NA < 0.02 91 1 0.107 0.0829 60 87 < 0.01 0.013 NA 0.023 87 USA Gardner KS, 2009, (FG 72) Soil SiL

SC b 1 0.104 0.0727 01 140 < 0.01 < 0.01 NA < 0.02 89 RAISP006 IS014-09HA d

1 0.106 0.0746 13 114 < 0.01 < 0.01 NA < 0.02 90 d 1 0.105 0.0755 60 91 < 0.01 < 0.01 NA < 0.02 89 d SC a, b 1 0.104 0.0754 01 81 < 0.01 < 0.01 NA < 0.02 92 RAISP01

0 IS041-09HA

1 0.104 0.0732 60 8181 < 0.01 < 0.01 NA < 0.02 92 USA Carlyle IL, 2009,

SC b 1 0.105 0.0528 00 130 < 0.01 < 0.01 NA < 0.02 87 RAISP006 IS016-

Isoxaflutole

1248



DAT IFT, mg/kg

IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg eq

% DM


(FG 72) Soil SiL

09HA

1 0.104 0.0433 14 104 < 0.01 0.013 NA 0.023 88 1 0.104 0.0743 51 97 < 0.01 0.027 NA 0.037 87 USA Arkansaw WI, 2009, (FG 72) Soil SaL

SC b 1 0.106 0.0564 00 144 < 0.01 < 0.01 NA < 0.02 84 RAISP006 IS017-09HA

1 0.105 0.0559 15 110 < 0.01 < 0.01 NA < 0.02 80 1 0.106 0.0558 60 82 < 0.01 0.011 NA 0.021 85 USA Northwood ND, 2009, (FG 72) Soil SiL

SC b 1 0.104 0.0553 NS 147 < 0.01 < 0.01 NA < 0.02 80 RAISP006 IS018-09HA

1 0.105 0.0561 15 110 < 0.01 < 0.01 NA < 0.02 80 1 0.107 0.0566 60 97 < 0.01 < 0.01 NA < 0.02 84 USA Sheridan IN,2009, (FG 72) Soil SiL

SC b 1 0.102 0.0554 00 133 < 0.01 < 0.01 NA < 0.02 85 RAISP006 IS021-09HA

1 0.106 0.0599 15 104 < 0.01 < 0.01 NA < 0.02 85 1 0.103 0.0595 60 94 < 0.01 < 0.01 NA < 0.02 85 USA Dudley MO, 2009, (FG 72) Soil SiL


1 0.106 0.0567 60 80 < 0.01 0.019 NA 0.029 86

Soil LSa=loamy sand, SiL = silty loam, CL=clay loam, SaL=sandy loam, L=loam, SiCL=silty clay loam, SaCL=sandy clay loam GS growth stage, according to BBCH, NS not stated, NA = not analysed Total = sum of IFT and IFT-DKN, expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. a spray mix prepared from SC 480 g/L isoxaflutole and SC 480 g/L of the isopropylammonium salt of glyphosate (see introductory text) b Additionally, adjuvants were used, which included AMS at 2% w/w, a non-ionic surfactant ranging from 0.5% to 1% v/v, a drift control agent, and if necessary anti-foam. c Sample sizes were below the required minimum of 1 kg (in accordance with FAO manual 2009): (200–440 g). Samples are considered not representative and trials cannot be selected for MRL derivation, if according to cGAP. d Seeds were planted in an area that received isoxaflutole in the previous growing season. However, this did not affect the results, since no isoxaflutole residues were found in the control samples (< LOQ).

Maize (grains)

Supervised trials on maize were conducted in Spain, Italy, Greece, Portugal, Germany, France, Netherlands and the United Kingdom in 2005 and 2006 and in USA and Canada in 1998, 2005 and 2006 [Zimmer and Wieland, 2007, M-282674-01-1, report RA-2587/05; Wolters and Erler, 2007b, M-284423-01-1, report RA-2510/06; Wolters, 2007b, M-285014-01-1, report RA-2615/06; Zimmer, 2006, M-281611-01-1, report RA-2588/05; Wolters and Erler, 2007a, M-284416-01-1, report RA-2511/06; Wolters, 2007a, M-285005-01-1, report RA-2616/06, Mickelson, 1999a, M-240663-01-1, report AA98714495; Fischer and Helfrich, 2007, M-285889-01-1, report RAUBP008; Vaughn and

Isoxaflutole

1249

Cosgrove, 2007, M-286216-02-1, report RAUBP011]. Small plots of maize (50–1700 m2) were treated as indicated in Table 49 using knapsack, tractor/ATV mounted or wheelbarrow sprayers with spray volumes ranging from 90–300 L/ha


� a single bare ground application of isoxaflutole (pre-emergence of the crop) � a single application of isoxaflutole in combination with the safener cyprosulfamide at BBCH

12–14, � a single application of isoxaflutole in combination with thiencarbazone-methyl and the

safener cyprosulfamide at BBCH 13–14. � a single bare ground application of isoxaflutole in combination with the safener

cyprosulfamide (pre-emergence of the crop) followed by a second application with thiencarbazone-methyl in combination with the safener cyprosulfamide at BBCH 18.

Field samples of maize grains were taken as: “immature kernel” (milk ripeness, BBCH 79, sweetcorn kernel) or “field corn” (earliest commercial harvest, mature, BBCH 89). Single field samples were taken in the EU trials, duplicate field samples were taken in the USA trials. All sample sizes were at least 1 kg (in accordance with FAO manual 2009). All samples were stored frozen with a maximum storage period of 244 days at -18 ºC (EU trials), 185 days at -10 ºC (AA987144495) or 544 days at -15 ºC (RAUBP008), 139 days at -20 ºC (Canadian trials).

In the EU trials, isoxaflutole and its metabolites IFT-DKN and IFT-BA were determined using HPLC-MS-MS method 00985/M001. This method is considered valid in the range 0.01–0.1 mg/kg in maize grains. Average concurrent recoveries at 0.01–5.0 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (LOQ) for each analyte. Average concentrations for single field samples are shown in Table 49.

In the 1998 US trials, isoxaflutole and its metabolites IFT-DKN and IFT-BA were determined using HPLC-MS-MS method CAL#019-03 revision 1. This method is considered valid in the range 0.01–0.10 mg/kg in maize grains. Individual concurrent recoveries at 0.01, 0.1 and 10 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 49.

In the 2005 and 2006 Canadian and US trials, isoxaflutole and its metabolite IFT-DKN and IFT-BA were determined using modification C of HPLC-MS-MS method CAL#019-03. Modification C is considered valid in the range 0.01–0.2 mg/kg for IFT, 0.02–0.3 mg/kg for IFT-DKN and 0.03–0.3 mg/kg for IFT-BA in maize grains. Average concurrent recoveries at 0.01, 0.05 and 0.2 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (reported LOQ). Average concentrations for duplicate field samples are shown in Table 49. Because of validation results, the LOQs were increased to 0.02 mg/kg for IFT-DKN and 0.03 mg/kg for IFT-BA (indicated by note f).

Note: Storage stability studies on commodities with high starch content showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 15 months. Storage stability studies on various commodities showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 12 months but converts to IFT-BA within 23 months of storage. Since maize grains have been stored for longer than 3 months, part of the IFT may have degraded to IFT-DKN. Except for trials in report RAUBP008, maize grains have been stored for less than 12 months and these results are considered reliable. Samples from report RAUBP008 have been stored for longer than 12 months and part of the IFT-DKN may have degraded to IFT-BA. However, IFT, IFT-DKN and IFT-BA are all below LOQ and degradation remains unnoticed. Therefore, these results are considered reliable as well.

Isoxaflutole

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Table 49 Isoxaflutole related residues after pre- or post-emergent treatment in maize grains

MAIZE Location, year, (variety)

FL no kg ai/ha kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial


SC a 1 0.10 0.034 13 79 g 89

78 127

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05; R 2005 0623/6


SC b 1 0.099 0.033 13 79 g 89

77 135

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2510/06 R 2006 0795/4

Germany Euskirchen - Kessenich (Nordrhein-Westfalen) (Egrin FAO 220), Soil: SiC

SC a 1 0.10 0.034 13 79 g 89

90 128

< 0.01 < 0.01

< 0.01 < 0.01

0.01 0.01

< 0.02 < 0.02

RA-2587/05 R 2005 0961/8

Germany Leverkusen (Nordrhein-Westfalen), 2006 (Bunguy); Soil: SaL

SC c 1 c 0.10 0.034 05 79 g 89

91 140

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0801/2


SC c 1 c 0.10 0.034 06 79 g 89

116 167

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0802/0


SC a 1 0.10 0.034 13 79 g 89

103 148

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05 R 2005 0959/6


SC b 1 0.099 0.033 14 79 g 89

93 123

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2510/06 R 2006 0796/2


SC c 1 c 0.10 0.034 07 79 g 85

101 124

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0800/4

Netherlands SC a 1 0.10 0.034 13 79 g 116 < 0.01 < 0.01 < 0.01 < 0.02 RA-

Isoxaflutole

1251



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

Zwaagdijk-Oost (Noord-Holland) 2005, (Rosalie), Soil: C

89 143 < 0.01 < 0.01 < 0.01 < 0.02 2587/05 R 2005 0962/6


SC a 1 0.10 0.034 14 79 g 89

71 127

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2587/05 R 2005 0958/8


SC b 1 0.099 0.033 13 79 g 89

96 139

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2510/06 R 2006 0073/9

SC c 1 c 0.10 0.034 01 79 g 89

120 163

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0627/3


SC c 1 c 0.10 0.034 00 79 g 89

112 179

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0799/7


SC a

1 0.10 0.034 13 79 g 89

83 148

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0624/4

S-France Vouille (Poitou-Charentes) 2006, (dkc 4845) Soil: SiC

SC b 1 0.099 0.033 13 79 g 89

98 153

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2511/06 R 2006 0074/7


SC c 1 c 0.10 0.034 06 79 g 89

105 170

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0628/1


SC c 1 c 0.10 0.034 01 79 g 89

91 128

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0803/9

Spain Vila-sacra (Cataluña)

SC a 1 0.10 0.034 13 79 g 89

84 141

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005

Isoxaflutole

1252



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

2005, (DKc6575); Soil: SaL

0963/4


SC b 1 0.099 0.033 14 79 g 89

67 132

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2511/06 R 2006 0798/9

SC c 1 c 0.10 0.034 07 79 g 89

89 154

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0804/7

Spain Alginet (Comuni dad Valen ciana) 2006, (Constan za) Soil: SiC

SC c 1 c 0.10

0.034 00 79 g 89

78 128

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0806/3

Italy San Carlo (FE) Emilia-Romagna 2005, (DK 440); Soil: SaC

SC a 1 0.10 0.034 13 79 g 89

79 128

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0964/2

Italy San Carlo (FE) Emilia-Romagna, 2006, (PR34N43) Soil: SaC

SC b 1 0.099 0.033 13 79 g 89

75 124

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2511/06 R 2006 0797/0

SC c 1 c 0.10 0.034 01 79 g 89

100 149

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0805/5


SC a 1 0.10 0.034 13 79 g 89

77 137

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 0.02

< 0.02 < 0.02

RA-2588/05 R 2005 0965/0


SC a 1 0.10 0.034 13 79 g 89

80 133

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005 0966/9

USA Lehigh PA, 1998 (Mycogen 2598) Soil: SiL

WG 1 0.16 0.089 bare soil

Mat 143 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-01

USA Wilson NC, 1998, (Pioneer


Mat 133 < 0.01 < 0.01 0.041 < 0.02 AA98714495 14495-

Isoxaflutole

1253



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

3163) Soil: SaL

02

USA Greene IA, 1998, (Dekalb DK 561SR) Soil: L


Mat 139 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-04

USA Percival IA, 2005, (Sucrossco 2009) Soil: SiL

SC a 1 0.13 0.10 12 ECH 125 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP008, UB086-05H

USA Jefferson IA, 2005 (DKC63-81) Soil: L


USA Shelby MO, 1998, (Novartis NK 6800) Soil: L


Mat 132 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-05

Soil: SiL WG 1 0.16 0.090 bare soil

Mat 140 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-21

USA Stoddard MO, 1998, (Pioneer 3394) Soil: SiL


Mat 115 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-10

USA Butler MO, 2005, (Garst 8287 RR) Soil: SiCL


USA Clarence MO, 2005, (Pioneer 35P12) Soil: CL


USA Fayette OH, 1998, (SC 1096) Soil: SiCL


Mat 153 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-06

(SC 1068) Soil: SiL


22% Dent 19% 19% 19% moisture

124 131 138 145 152

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02 < 0.02

AA98714495 14495-19

USA Marysvillle

SC a 1 0.13 0.079 12 ECH 108 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP008,

Isoxaflutole

1254



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

OH, 2005, (Shur Grow SG-751) Soil: CL

UB089-05H

USA New Holland OH, 2005 (Crows 7R321) Soil: L


USA Clay SD, 1998, (Dekalb DK 493RR) Soil: L


Mat 143 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-07

USA Yankton SD, 1998 (Dekalb, DK 493RR) Soil: SiCL


Mat 144 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-08

USA Grand Forks ND, 1998, (Mycogen 2110 R3) Soil: SiL


Mat 146 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-09

USA Steele ND, 1998, (Mycogen 2110 R3) Soil: SiL


Mat 138 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-16

USA Northwood ND, 2005, (39D81) Soil: L


USA Stark IL, 1998 (Pioneer 3394) Soil: SiL


Mat 142 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-11

USA Bond IL, 1998, (Pioneer 3394) Soil: SiL


Mat 111 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-12

USA Clinton IL, 1998, (Pioneer 33V08) Soil: SiL


R6 134 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-20

USA Seymour IL, 2005 (Garst 8550) Soil: SiL

SC a 1 0.13 0.10 12 ECH 129 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP008, UB082-05D

Isoxaflutole

1255



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

USA Monticello IL, 2005 (Pioneer 37H26) Soil: SiL

SC a 1 0.13 0.087 12 ECH 102 < 0.01 < 0.02f 0.030 < 0.03f RAUBP008, UB083-05H

USA Carlyle IL, 2005 (FS 6455 RR/YGCB) Soil: SiL


USA York NE, 1998, (Northrup King N3030BT) Soil: SiL


Mat 106 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-13


32% 25% 17% 15% 15% moisture

103 109 116 123 130

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02 < 0.02

AA98714495 14495-18

USA Polk NE, 1998, (Northrup King N3030BT) Soil: LSa


Mat 109 < 0.01 < 0.01 0.013 < 0.02 AA98714495 14495-14

USA Springfield NE, 2005 (NK70T9) Soil: SiL


USA Stafford KS, 1998, (Pioneer 3737) Soil: LSa


Mat 103 < 0.01 < 0.01 0.012 < 0.02 AA98714495 14495-15

USA Stilwell KS, 2006, (Garst 8478 LL/CB/GT) Soil: SiL

SC a 1 0.14 0.10 12 ECH 119 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP008, UB080-05DA d

USA Clay TX, 1998, (Northrup King 512RR) Soil: Sa


Mat 104 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-17

USA East Bernard TX, 2005 (DKC69-71) Soil: SaL


USA SC a 1 0.13 0.098 12 ECH 103 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP

Isoxaflutole

1256



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

Bumpass VA, 2005, (Dekalb) Soil: SaCL

008, UB078-05H

USA Tifton GA, 2005 (Pioneer 31N26) Soil: SaL


USA Gosport IN, 2005, (Pioneer 34D71) Soil: SiL


USA Sheridan IN, 2005, (Golden Harvest RR) Soil: SiL


USA Sabin MN, 2005, (Pioneer 39H85) Soil: Si


USA Campbell MN, 2005, (Croplan 212RR2) Soil: CL


USA Arkansaw WI, 2005, (Dekalb DKC 46-28 RR) Soil: SaL


Canada Clarksburg, ON, 2006, (Pioneer 39F27) Soil: L

SC a 1 0.21 0.11 13 – 114 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 01-06H

Canada Metz, ON, 2006 (Pioneer 39H86) Soil: L

SC a 1 0.21 0.11 12-13 – 112 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 02-06H

Canada Rockwood, ON, 2006 (Pioneer 38K35) Soil: L

SC a 1 0.21 0.11 13 – 96 103 110 117

< 0.01 < 0.01 < 0.01 < 0.01

< 0.02f < 0.02f

< 0.02f

< 0.02f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

RAUBP011, 03-06D

Canada Breslau, ON, 2006,

SC a 1 0.21 0.11 13 – 110 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 04-06H

Isoxaflutole

1257



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

(Pioneer 39F28) Soil: SaL Canada Greenfield, ON, 2006, (Pioneer 39F28) Soil: SaL

SC a 1 0.21 0.11 13 – 110 < 0.01 < 0.02f 0.036 < 0.03f RAUBP011, 05-06H

Canada Woodstock, ON, 2006, (NK N29G7) Soil: L

SC a 1 0.22 0.11 13 – 110 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 06-06H

Canada Port Burwell, ON, 2006, (Pioneer 38A24) Soil: SaL

SC a 1 0.21 0.11 13 – 113 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 07-06H

Canada Paris, ON, 2006 (Dekalb 4057) Soil: SaL

SC a 1 0.21 0.11 12–13 – 108 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 10-06H

Canada Springford, ON, 2006 (DKC42-70) Soil: SaL

SC a 1 0.22 0.11 12–13 – 110 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 11-06H

Canada Branchton, ON, 2006 (Hybrid Corn 38-B84) Soil: SiL

SC a 1 0.22 0.11 12–13 – 96 103 110 117

< 0.01 < 0.01 < 0.01 < 0.01

< 0.02f < 0.02f

< 0.02f

< 0.02f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

< 0.03f

RAUBP011, 12-06D

Canada Pike River, QC, 2006, (ZP172) Soil: L

SC a 1 0.23 0.11 13 – 111 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 08-06H

Canada St-Pie-de-Bagot, QC, 2006, (ZP172) Soil: LSa

SC a 1 0.21 0.10 13 – 111 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 09-06H

Canada St-Marc-sur-Richelieu, QC, 2006, (Pioneer RR38P09) Soil: CL

SC a 1 0.21 0.12 13 – 111 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 13-06H

Canada Acton Vale, QC, 2006, (NK N16-N7) Soil: Sa

SC a 1 0.22 0.13 13 – 110 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP011, 14-06H

Canada SC a 1 0.21 0.12 13 – 111 < 0.01 < 0.02f < 0.03f < 0.03f RAUBP

Isoxaflutole

1258



GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

Ste-Madeleine, QC, 2006, (Mycogen 2P172) Soil: SaL

011, 15-06H

Soil:C = Clay, L= loam, Si = silt; Sa= sand; CL = clay loam; CSa = clay sand, CSi= clay silt; LSa = loamy sand; SiC = silty clay; SaCL=sandy clay loam; SiCL = silty clay loam, SiL = silty loam; SaC = sandy clay; SaL = sandy loam, SaSi = sandy silt; GS Mat = mature, R6 = reproductive stage 6, equivalent to BBCH 89, ECH = earliest commercial harvest Total = sum of IFT and IFT-DKN, expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. a SC formulation contained 20.4% w/w isoxaflutole and 20.3% w/w cyprosulfamide (safener); or 240 g/L each b SC formulation contained 225 g/L isoxaflutole, 150 g/L cyprosulfamide (safener) and 90 g/L thiencarbazone-methyl c A first spray application was performed with an SC formulation (containing 240 g/L isoxaflutole and 240 g/L cyprosulfamide (safener)) at the pre-emergence stage followed by a second application with an SC formulation (containing 225 g/L thiencarbazone-methyl and 225 g/L cyprosulfamide (safener)) at growth stage BBCH 18. Days after last treatment (DAT) are related to the isoxaflutole treatment (i.e. days after the first treatment). d Trial UB080-05D was repeated in 2006 as UB080-05DA due to improper application timing in the original trial. e Individual results for IFT and IFT-DKN not considered reliable, because samples have been stored for longer than 12 months. f LOQ for IFT-DKN needs to be increased to 0.02 mg/kg and LOQ for IFT-BA needs to be increased to 0.03 mg/kg. This increase has been taken into account in total residue calculation. g kernel immature (milk ripe)

Sugar cane (billets)

Supervised trials on sugarcane were conducted in Australia during the 1996–1998 growing seasons [Davis and Keats, 1999a, M-284383-01-1, Report AK99009; Davis and Keats, 1999b, M-284347-01-1, Report AK99010; Davis and Keats, 1999c, M-284356-01-1, Report AK99011; Davis and Keats, 1999d, M-284369-01-1, Report AK99012; Davis and Keats, 1999e, M-284408-01-1, Report AK99017], in Mexico during the 1999 growing season [Gough, 2000, M-238729-01-1, Report 99717930] and in Brazil during the 1998-1999 growing season [Tornisielo, 2000b, M-287041-01-1 Report USP BRA98R27-1; Tornisielo, 2000a, M-287038-01-1 Report USP BRA98R27-2].

In Australia, small plots of sugar cane (15–40 m2) were treated as indicated in Table 50 using plot boom sprayers or spray bars with spray volumes ranging from 238–265 L/ha. In two parallel plots sugarcanes in the 5–8 leaf stage received a single soil application of isoxaflutole at two different application rates.

In Mexico, small plots of sugar cane (210–216 m2) were treated as indicated in Table 50 using commercial ground equipment for broadcast application with spray volumes ranging from 252–411 L/ha. The application was made at the regrowth stage, up to 4 months after harvest of the previous crop, and about 8–10 months before expected crop maturity.

In Brazil, small plots of sugar cane (72–78 m2) were treated as indicated in Table 50 using backpack sprayers with spray volumes of 200 L/ha. Sugarcane was treated twice with isoxaflutole. The first application was performed at pre-emergence and the second one after emergence. In parallel plots, different application rates were tested.

Field samples of whole canes were collected at a harvestable maturity: single field samples in Australia: replicate field samples in Mexico. Samples from each plot were taken from at least 15 whole canes randomly from the entire plot (in accordance with the FAO 2009 manual). The top 30 cm of the whole canes were then cut off using secateurs to provide forage (tops) and cane. The remaining cane was cut into billets of 20–25 cm in length. Equal portions of tops, middle and bottom were

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sampled from at least 12 stalks to provide at least 1 kg of whole cane billets. All samples were stored frozen for a maximum storage period of 33 days at -10 ºC (Mexican trials), 5 months at -20 ºC (AK 99009, AK 99010) or 10 months at -20 ºC (AK 99011, AK 99012) or 20 months at -20 ºC (AK 99017), 24 months at -10 ºC (Brazilian trials).

In the Australian trials, samples were analysed using GC-MS-MS modification A of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and total residues are expressed as isoxaflutole equivalents. This method is considered valid at 0.01 mg/kg in sugar cane billets. Average concurrent recoveries at 0.01 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (LOQ). Average results of the replicate field samples are reported in Table 50.

In the Mexican trials, samples were analysed using GC-MS modification D of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and total residues are expressed as isoxaflutole equivalents. This method is considered valid in the range 0.01–0.02 mg/kg in sugar cane billets. Average concurrent recoveries at 0.01, 0.02, 0.1 mg/kg analyte were within 70–120% limits (n=1–2 each), except for IFT at 0.01 mg/kg (128%, n=2) and IFT-DKN at 0.01 mg/kg (122%, n=2). Levels in control samples were < 0.01 mg/kg (LOQ). Average results of the replicate field samples are reported in Table 50.

In the Brazilian trials, samples were analysed using GC-MS-MS modification C of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and total residues are expressed as isoxaflutole equivalents. This method is considered valid in the range 0.01–0.02 mg/kg in sugar cane billets. Average concurrent recoveries at 0.01 and 0.02 mg/kg were within 70–120% for IFT. Recoveries for other metabolites were not verified. Levels in control samples were < 0.01 mg/kg (LOQ). Average results of the replicate field samples are reported in Table 50.

Note: Storage stability studies on commodities with high water content showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 20 months. Since sugarcanes have been stored for 20 months or shorter, all results are considered reliable.

Table 50 Isoxaflutole related residues after pre-harvest soil directed treatment in sugarcane (billets i.e. raw canes)

SUGAR CANE Location, year, (variety)

FL No (in ter val)

kg ai/ha kg ai/hL GS last appl

GS harv

DAT IFT mg/kg

RPA 202 248 mg/kg

RPA 203 328 mg/kg

Total mg/kg eq

Study, Trial

Australia Tolga Mareeba, QLD, 1998, (Q 120) Soil: SaCL

WG 1 0.150 0.057 120 cm CH 166 NA NA NA < 0.01 AK99009

WG 1 0.225 0.085 120 cm CH 166 NA NA NA < 0.01 Australia Atherton, QLD, 1998, (Q 124) Soil: CL

WG 1 0.150 0.057 120 cm CH 133 NA NA NA < 0.01 AK99010

WG 1 0.225 0.087 120 cm CH 133 NA NA NA < 0.01 Australia Murwillumbah QLD, 1997/98, (Q 170) Soil: peat

WG 1 0.150 0.058 110 cm (30 cm TVD)

CH 167 NA NA NA < 0.01 AK99011

WG 1 0.225 0.086 110 cm (30 cm

CH 167 NA NA NA < 0.01

Isoxaflutole

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SUGAR CANE Location, year, (variety)

FL No (in ter val)

kg ai/ha kg ai/hL GS last appl

GS harv

DAT IFT mg/kg

RPA 202 248 mg/kg

RPA 203 328 mg/kg

Total mg/kg eq

Study, Trial

TVD) Australia Rocky Point, QLD, 1997/98 (Q 154) Soil: peat

WG 1 0.150 0.058 100 cm (30 cm TVD)

CH 193 NA NA NA < 0.01 AK99012

WG 1 0.225 0.086 100 cm (30 cm TVD)

CH 193 NA NA NA < 0.01

Australia Jardine QLD, 1996 (Q 96) Soil: baratter cracking C

WG c 1 0.150 0.063 5–7 leaves (25–30 cm TVD)

CH 206

NA NA NA < 0.01 AK99017

WG c 1 0.300 0.126 5–7 leaves (25–30 cm TVD)

CH 206

NA NA NA < 0.01

Mexico Cordoba, Veracruz, 1999-2000 (Mex 69-290) Soil: SaL

WG 1 0.123

0.030 38 cm re-growth a

CH 258 NA NA NA < 0.01 99717930, 17930-01

Mexico Ahualulco de Mercado, Jalisco, 1999-2000 (Mex 57-473) Soil: SaL

WG 1 0.121


CH 255 NA NA NA < 0.01 99717930, 17930-02

Mexico Tres Valles, Veracruz, 1999-2000 (Mex 69-290) Soil: SaL

WG 1 0.120


CH 257 NA NA NA < 0.01 99717930, 17930-03

Brazil Cosmopolis Sao Paulo, 1999-2000 (RB-72-454) Soil: C

WG 2 (92)

0.075 0.075

0.038 0.038

b – 95 NA NA NA < 0.01 USP BRA98R27-1 trial A

WG 2 (92)

0.150 0.150

0.075 0.075

b – 95 NA NA NA < 0.01 USP BRA98R27-1 trial B

Brazil Assis, Sao Paulo, 1998 (RB-79-1011) Soil: medium C

WG 2 (61)

0.075 0.075

0.038 0.038

b – 92 NA NA NA < 0.01 USP BRA98R27-2 trial A

WG 2 (61)

0.15 0.15

0.075 0.075

b – 92 NA NA NA < 0.01 USP BRA98R27-2 trial B

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Soil: C = clay, CL = clay loam, SaCL = sandy clay loam, SaL = sandy loam Total = Total residues of IFT = measured sum of IFT, RPA 20248 and IFT-BA residues, expressed as isoxaflutole equivalents. The total residue is determined via a common moiety method which converts isoxaflutole, IFT-DKN and IFT-BA onto the derivative RPA 204497 TVD = top visible dewlap CH = commercial harvest (normal crop maturity, marketable specimens) a regrowth stage: up to 4 months after harvest of the previous crop b first application in pre-emergence followed by a second application post-emergence c X-77 wetting agent (250 ml/hL) was added

Poppy seed

Supervised trials on poppy were conducted in France, Spain, Germany, Netherlands and Hungary in 2008 and 2009 [Billian and Reinecke, 2010, M-360144-02-1, Report 08-2095; Billian and Krusell, 2010b, M-360114-02-1, Report 08-2094; Billian and Krusell, 2010a, M-364166-01-1, Report 09-2085; Noss and Reinecke, 2010, M-366025-01-1, report 09-2022]. Small plots of poppy (45–910 m2) were treated as indicated in Table 51 using knapsack or tractor mounted boom sprayers with spray volumes ranging from 300–400 L/ha. Poppy plants were treated with a single application of isoxaflutole in combination with the safener cyprosulfamide at pre-emergence stage of the crop.

In some trials phytotoxicity signs were seen a few weeks after treatment: dead plants, plants with burning damage or symptoms of discoloration, plants with reduced growth, reduced emergence of plants (trial 08-2094-02&03&04, trial 08-2095/02&04). These phytotoxicity problems affected the yield of the poppy seeds.

Single field samples of seed were taken at harvest (BBCH 89). All sample sizes were at least 1 kg (in accordance with FAO manual 2009), except in trials 09-2022/01&02 and 08-2095/02&04 (0.1–0.7 kg) as indicated in Table 51. All samples were stored frozen at –18 ºC with a maximum storage period of 111 days (report 0902085), 134 days (report 09-2022) or 447 days (report 08-2094 and 08-2095).

Isoxaflutole and its metabolites IFT-DKN and IFT-BA were determined using HPLC-MS-MS method 00985/M001. This method is considered valid in the range 0.01–0.10 mg/kg in poppy seeds. Average concurrent recoveries at 0.01–0.1 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (LOQ) for each analyte. Average concentrations for single field samples are shown in Table 51.

Note: Seeds need to be grinded prior to analyses, since residue levels may increase upon grinding. At first instance seeds were not grinded prior to extraction. In those cases, where residues of IFT-BA were seen, the analysis was repeated by grinding the seeds prior to extraction. All results are therefore considered reliable.

Note: Storage stability studies on various commodities showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 11–20 months. Storage stability studies on commodities with high oil content showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 16 months but converts to IFT-BA within 23 months of storage. Since poppy seeds have been stored for longer than 3 months, part of the IFT may have degraded to IFT-DKN. Since poppy seeds have been stored for less than 16 months, levels of IFT-DKN are not further degraded to IFT-BA and can be considered reliable. If the residue definition includes both IFT and IFT-DKN, residues can be taken for MRL derivation, if according to cGAP.

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Table 51 Isoxaflutole related residues after pre-emergent treatment in poppy seeds

POPPY Location, year, (variety)

FL No kg ai/ha kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

Total mg/kg eq

Study, Trial

Netherlands Beenster 2008, (Pulchinella) Soil: C

SC a 1 0.10 0.034 00 89 100 < 0.01 < 0.01 < 0.01 < 0.02 08-2094-trial 02 b

Germany Burscheid, 2008 (Mieszko) Soil: CSi

SC a 1 0.10 0.034 00 89 130 < 0.01 < 0.01 < 0.01 < 0.02 08-2094-trial 03 b

Hungary Soponya Fejér 2009, (Korona) Soil: SaL

SC a 1 0.10 0.034 00 pre-em

89 129 < 0.01 < 0.01 < 0.01 < 0.02 09-2022-trial 01 c d

Hungary Arpadhalom Csongrád 2009, (Korona) Soil: SaL

SC a 1 0.10 0.034 00 89 119 < 0.01 < 0.01 < 0.01 < 0.02 09-2022-trial 02 c d

N-France Chilly, 2008, (Miezko) Soil: SiL

SC a 1 0.10 0.034 00 89 126 < 0.01 < 0.01 < 0.01 < 0.02 08-2094-trial 04 b

S-France Boe 2008, (Mieszko) Soil: CSi

SC a 1 0.079 0.020 00 89 110 < 0.01 < 0.01 < 0.01 < 0.02 08-2095-trial 02 b d

S-France Chazay 2008, (Mieszko) Soil: L

SC a 1 0.079 0.026 00 89 110 < 0.01 < 0.01 0.02 < 0.02 08-2095-trial 04 c d

Spain Minaya 2008, (Luisiana) Soil: C

SC a 1 0.079 0.026 03 89 119 < 0.01 < 0.01 < 0.01 < 0.02 08-2095-trial 01 b

Spain Minaya 2009 (Luisiana) Soil: C

SC a 1 0.079 0.026 00 89 140 < 0.01 < 0.01 < 0.01 < 0.02 09-2085-trial 02 b, d

Spain Albacete 2008, (Luisiana) Soil: C

SC a 1 0.079 0.026 03 89 128 < 0.01 < 0.01 < 0.01 < 0.02 08-2095-trial 03 b

Isoxaflutole

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POPPY Location, year, (variety)

FL No kg ai/ha kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

Total mg/kg eq

Study, Trial

Spain La Herrera 2009, (Luisiana) Soil: SaL

SC a 1 0.079 0.026 00 89 133 < 0.01 < 0.01 < 0.01 < 0.02 09-2085-trial 01 c

Spain Pozohondo 2009 (Luisiana) Soil: SaL

SC a 1 0.079 0.026 00 89 132 < 0.01 < 0.01 < 0.01 < 0.02 09-2085-trial 03 b, d

Spain Tinarejos 2009, (Luisiana) Soil: SaL

SC a 1 0.079 0.026 00 89 125 < 0.01 < 0.01 < 0.01 < 0.02 09-2085-trial 04 b

Soil:C = Clay, L= loam, Si = silt; Sa= sand; CL = clay loam; CSa = clay sand, CSi= clay silt; LSa = loamy sand; SiC = silty clay; SaCL=sandy clay loam; SiCL = silty clay loam, SiL = silty loam; SaC = sandy clay; SaL = sandy loam, SaSi = sandy silt; total = sum of IFT and IFT-DKN, expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. a SC formulation contained 20.4% w/w isoxaflutole and 20.3% w/w cyprosulfamide (safener); or 240 g/L each b Before analysis, the seeds were not grinded. c Before analyses, the seeds were grinded/homogenised for 30 sec using a basic mixer. Grinding the seeds leads to residues of IFT-BA about a factor 2 higher compared to non-grinded seeds. d Sample sizes were less than the required minimum of 1 kg (in accordance with FAO manual 2009): 0.1–0.9 kg. In plot 082095-02&04, the reduced sample size is due to phytotoxicity effects: bad growing and bad conditions of plants. Samples are considered not representative and trials cannot be selected for MRL derivation, if according to cGAP.

Legume animal feeds

Supervised residue trials were conducted on chickpea forage and chickpea straw.

Chickpea forage


In 1999, different application scenarios were tested on parallel plots. Plots were treated once at different application rates and/or growth stages (before emergence of the crop or at 3 nodes stage). In 2011, four application scenarios were tested on parallel plots. Isoxaflutole was applied at rates of 0.075 kg ai/ha and 0.150 kg ai/ha to bare soil either as a single fallow application approximately four month before sowing the chickpea crops, or in conjunction with a second application at planting.

Replicate field samples of forage (or green material) were taken at pre-flowering (AK99056) or at 3–4 node stage (AQ reports) or 42–49 DAS (BCS-0370). Forage was sampled by hand with secateurs, cutting random samples of a chickpea plant down to 5 cm above ground level, from at least 10 separate plants from all over the plot. All sample sizes were less than the required minimum of 1 kg (in accordance with FAO manual 2009): 0.10 kg in report AK99056, 0.20 kg in BCS-0370,

Isoxaflutole

1264

0.25 kg in AQ00001 and AQ00003, 0.5 kg in AQ00002. Samples were stored frozen with a maximum storage period of 3–21 days at –20 ºC (AQ reports), 11 months at –20 ºC (AK99056) or 135 days at –9 ºC (BCS-0370).

In the trials from 1999, samples were analysed for total residues using GC-MS-MS modification B of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and total residues are expressed as isoxaflutole equivalents. This method is considered valid in the range 0.01–0.1 mg/kg in chickpea forage. Since total residue levels in the supervised trials ranged from 0.01–0.19 mg/kg, validation at higher levels is desirable. Average concurrent recoveries at 0.01 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 52.

In the trials from 2011, samples were analysed for IFT, IFT-DKN and IFT-BA using modification A of HPLC-MS-MS method ATM-0054.01. This method is considered valid in the range 0.01–0.1 mg/kg in chickpea forage. Since IFT-BA levels in the supervised trials ranged from 0.01–0.55 mg/kg, validation at higher levels is desirable. Average concurrent recoveries at 0.01–1.0 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 52.


Note: Storage stability studies on commodities with high water content showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 20 months. Storage stability studies on commodities with high water content showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 12 months but converts to IFT-BA within 23 months of storage. Since chickpea forages have been stored for a maximum of 11 months in the trials where a common moiety method was used and chickpea forages have been stored for less than 3 months in trials where individual compounds have been measured, all results are considered reliable.

Table 52 Isoxaflutole related residues after pre-or post-emergent treatment of chickpeas (forage or green material)

CHICK PEA FORAGE Location, year, (variety)

FL No & interval

kg ai/ha

kg ai/hL

timing DAT IFT mg/kg

IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg

report, trial

Australia Biniguy NSW, 2009, (Amethyst) Soil: brown Clay Loam


70 NA NA NA < 0.01 a AK99056 99NST01-A

c


70 NA NA NA 0.034 a

AK99056 99NST01-B

c Australia Horsham VIC, 2009, (Sona) Soil: Wimmera gray clay


30 NA NA NA 0.028 a

AQ00001 99h011-A c


30 NA NA NA 0.16 a

AQ00001 99h011-B c

WG d

1 0.056 0.075 Early Post-emergent

46 NA NA NA 0.040 a

AQ00002 99h015-A c

Isoxaflutole

1265


FL No & interval

kg ai/ha

kg ai/hL


IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg

report, trial

(3 nodes) WG

d 1 0.075 0.100 Early Post-

emergent (3 nodes)

46 NA NA NA 0.12 a

AQ00002 99h015-B c

WG d

1 0.094 0.13


46 NA NA NA 0.16 a

AQ00002 99h015-C c

WG e


46 NA NA NA 0.085 a

AQ00002 99h015-D c

WG e


46 NA NA NA 0.11 a

AQ00002 99h015-E c

WG e


46 NA NA NA 0.11 a

AQ00002 99h015-F c


WG d

1 0.075 0.15 Post sow/Pre-emergent

28

NA NA NA 0.095 a

AQ00003 99h010-A c

WG d


28

NA NA NA 0.15 a

AQ00003 99h010-B c

WG d


28

NA NA NA 0.19 a

AQ00003 99h010-C c

WG e


28

NA NA NA 0.11 a

AQ00003 99h010-D c

WG e


28

NA NA NA 0.15 a

AQ00003 99h010-E c

WG e


28

NA NA NA 0.15 a

AQ00003 99h010-F c

Australia Northam WA, 2011, (Desi) Soil: sandy loam

WG 1 0.075 0.033

bare soil 121 days before sowing

163 169 177

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.20 0.16 0.12

< 0.02 < 0.02 < 0.02 b

BCS-0370, C601 c


163 169 177

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.32 0.28 0.22

< 0.02 < 0.02 < 0.02 b

c

WG 2 (121 days)

0.075 0.075

0.033 0.075


42 48 56

< 0.01 < 0.01 < 0.01

0.060 0.045 0.030

0.34 0.29 0.24

0.070 0.055 0.040 b

c

WG 2 (121 days)

0.15 0.15

0.066 0.150


42 48 56

< 0.01 < 0.01 < 0.01

0.075 0.050 0.065

0.55 0.47 0.44

0.085 0.060 0.075 b

c



168 175 182

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 b

BCS-0370, C602 c

WG 1 0.15 0.11 bare soil 168 < 0.01 < 0.01 < 0.01 < 0.02 c

Isoxaflutole

1266


FL No & interval

kg ai/ha

kg ai/hL


IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg

report, trial

127 days before sowing

175 182

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02 b

WG 2 (127 days)

0.075 0.075

0.055 0.057


41 48 55

< 0.01 < 0.01 < 0.01

0.025 0.015 < 0.01

0.28 0.28 0.15

0.035 0.025 < 0.02 b

c

WG 2 (127 days)

0.15 0.15

0.11-0.11


41 48 55

< 0.01 < 0.01 < 0.01

0.025 0.020 < 0.01

0.40 0.28 0.22

0.035 0.030 < 0.02 b

c

Australia Shepparton East VIC, 2011, (Kabul) Soil: clay loam


156 164 171

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 b

BCS-0370, C603 c


156 164 171

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.020 0.030 0.030

< 0.02 < 0.02 < 0.02 b

c

WG 2 (107 days)

0.075 0.072

0.075 0.075


49 57 64

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.10 0.12 0.11

< 0.02 < 0.02 < 0.02 b

c

WG 2 (107 days)

0.15 0.15


49 57 64

< 0.01 < 0.01 < 0.01

0.020 0.020 0.020

0.24 0.22 0.20

0.03 0.03 0.03 b

c

a Total residue obtained by common moiety method, whereby residues are converted into RPA204497 and expressed as isoxaflutole equivalent (residue includes IFT, IFT-DKN and IFT-BA) b Total residue calculated by summing up residues of IFT and IFT-DKN and expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. c All sample sizes were less than the required minimum of 1 kg (in accordance with FAO manual 2009): 0.10 kg in report AK99056, 0.20 kg in BCS-0370, 0.25 kg in AQ00001 and AQ00003, 0.5 kg in AQ00002. However, two samples were taken from each trial plot (each from at least 10 plants in the AK and AQ reports) and the results are the mean of both samples. Therefore results can be considered representative and trials can be selected for MRL derivation, if according to cGAP. d added adjuvant: 0.25% X-77 e added adjuvant: Uptake oil

Chickpea fodder


In 1999, different application scenarios were tested on parallel plots. Plots were treated once at different application rates and/or growth stages (before emergence of the crop or at 3 nodes stage).

In 2011, four application scenarios were tested on parallel plots. Isoxaflutole was applied at rates of 0.075 kg ai/ha and 0.150 kg ai/ha to bare soil either as a single fallow application

Isoxaflutole

1267

approximately four month before sowing the chickpea crops, or in conjunction with a second application at planting.

Replicate field samples of chickpea fodder were taken at maturity. In report AK99054, chickpea plots were mechanically harvested and fodder was randomly selected from the trash coming out of the back of the harvester. In report AK99056, BCS-370 and AQ reports fodder was sampled by hand with secateurs, cutting random samples of a dry chickpea plant down to 5 cm above ground level, from at least 10 separate plants from all over the plot. Sample sizes were at least 0.5 kg in report BCS-0370, AQ00003 and AK99054. Sample sizes from all other reports were below the required minimum of 0.5 kg (in accordance with FAO manual 2009): 0.1 kg in report AK99056, 0.25 kg in AQ00001 and AQ00002. Samples were stored frozen with a maximum storage period of 2–21 days at -20 ºC (AK99054 and AQ reports), or 11 months at -20 ºC (AK99056) or 25 days at -8 ºC (BCS-0370).

In the trials from 1999, samples were analysed for total residues using GC-MS-MS modification B of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and total residues are expressed as isoxaflutole equivalents. This method is considered valid in the range 0.01–0.1 mg/kg in chickpea straw. Average concurrent recoveries at 0.01 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 53.

In the trials from 2011, samples were analysed for isoxaflutole, IFT-DKN and IFT-BA using modification A of HPLC-MS-MS method ATM-0054.01. This method is considered valid in the range 0.01–0.1 mg/kg in chickpea straw. Since IFT-BA levels in chickpea straw ranged from < 0.01–0.12 mg/kg validation at higher levels is desirable. Average concurrent recoveries at 0.01–1.0 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 53.


Note: Storage stability studies on straw showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 15 months. Storage stability studies on various commodities showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 12 months but converts to IFT-BA within 23 months of storage. Since chickpea straw has been stored for a maximum of 11 months in the trials where a common moiety method was used and chickpea straw has been stored for less than 3 months in trials where individual compounds have been measured, all results are considered reliable.

Table 53 Isoxaflutole related residues after pre- or post-emergent treatment of chickpeas (straw)

CHICK PEA STRAW Location, year, (variety)

FL No & interval

kg ai/ha

kg ai/hL


IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg

% dm

Report, trial

Australia Pittsworth QLD, 2009 (Norwin) Soil: black earth


185 NA NA NA < 0.01 a AK99054 99NST26-A


185 NA NA NA < 0.01 a

AK99054 99NST26-B

Australia Biniguy NSW, 2009,


155 NA NA NA < 0.01 a AK99056 99NST01-A c

Isoxaflutole

1268


FL No & interval

kg ai/ha

kg ai/hL


IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg

% dm

Report, trial

(Amethyst) Soil: brown CL WG 1 0.15 0.25 3 days post

sowing 155 NA NA NA < 0.01 a AK99056

99NST01-B c

Australia Horsham VIC, 2009, (Sona) Soil: Wimmera gray clay


184 NA NA NA < 0.01 a AQ00001 99h011-A c


184 NA NA NA < 0.01 a AQ00001 99h011-B c

WG d 1 0.056 0.075 Early Post-emergent (3 nodes)

147 NA NA NA < 0.01 a AQ00002 99h015-A c

WG d 1 0.075 0.100 Early Post-emergent (3 nodes)

147 NA NA NA < 0.01 a AQ00002 99h015-B c

WG d 1 0.094 0.13


147 NA NA NA 0.012 a AQ00002 99h015-C c


147 NA NA NA < 0.01 a AQ00002 99h015-D c


147 NA NA NA < 0.01 a AQ00002 99h015-E c


147 NA NA NA < 0.01 a AQ00002 99h015-F c



188 NA NA NA < 0.01 a AQ00003 99h010-A


188 NA NA NA < 0.01 a AQ00003 99h010-B


188 NA NA NA 0.018 a AQ00003 99h010-C


188 NA NA NA < 0.01 a AQ00003 99h010-D


188 NA NA NA < 0.01 a AQ00003 99h010-E


188 NA NA NA < 0.01 a AQ00003 99h010-F

Australia Northam WA, 2011, (Desi)


295 < 0.01 < 0.01 0.030 < 0.02 b 59 BCS-0370, C601

Isoxaflutole

1269


FL No & interval

kg ai/ha

kg ai/hL


IFT-DKN, mg/kg

IFT-BA, mg/kg

Total, mg/kg

% dm

Report, trial

Soil: sandy loam WG 1 0.15 0.066 bare soil

121 days before sowing

295 < 0.01 < 0.01 0.055 < 0.02 b 59

WG 2 (121 days)

0.075 0.075

0.033 0.075


174 < 0.01 < 0.01 0.075 < 0.02 b 59

WG 2 (121 days)

0.15 0.15

0.066 0.150


174 < 0.01 < 0.01 0.12 < 0.02 b 59



284 < 0.01 < 0.01 < 0.01 < 0.02 b 42 BCS-0370, C602


284 < 0.01 < 0.01 < 0.01 < 0.02 b 42

WG 2 (127 days)

0.075 0.075

0.055 0.057


157 < 0.01 < 0.01 0.065 < 0.02 b 42

WG 2 (127 days)

0.15 0.15

0.11-0.11


157 < 0.01 < 0.01 0.030 < 0.02 b 42

Australia Shepparton East VIC, 2011, (Kabul) Soil: clay loam


324 < 0.01 < 0.01 < 0.01 < 0.02 b 27 BCS-0370, C603


324 < 0.01 < 0.01 < 0.01 < 0.02 b 27

WG 2 (107 days)

0.075 0.072

0.075 0.075


217 < 0.01 < 0.01 0.015 < 0.02 b 27

WG 2 (107 days)

0.15 0.15


217 < 0.01 < 0.01 0.060 < 0.02 b 27

a Total residue obtained by common moiety method, whereby residues are converted into RPA204497 and expressed as isoxaflutole equivalent (residue includes IFT, IFT-DKN and IFT-BA) b Total residue calculated by summing up residues of IFT and IFT-DKN and expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. c Sample sizes were below the required minimum of 0.5 kg (in accordance with FAO manual 2009): 0.1 kg in report AK99056, 0.25 kg in AQ00001 and AQ00002. However, two samples were taken from each trial plot (each from at least 10 plants in the AK and AQ reports) and the results are the mean of both samples. Therefore results can be considered representative and trials can be selected for MRL derivation, if according to cGAP. d added adjuvant: 0.25% X-77 e added adjuvant: Uptake oil

Isoxaflutole

1270

Forage and fodder of cereal grains and grasses

Supervised residue trials were available for maize forage and maize fodder.

Maize forage

Supervised trials on maize were conducted in Spain, Italy, Greece, Portugal, Germany, France, Netherlands and the United Kingdom in 2005 and 2006 and in USA and Canada in 1998, 2005 and 2006 [Zimmer and Wieland, 2007, M-282674-01-1, report RA-2587/05; Wolters and Erler, 2007b, M-284423-01-1, report RA-2510/06; Wolters, 2007b, M-285014-01-1, report RA-2615/06; Zimmer, 2006, M-281611-01-1, report RA-2588/05; Wolters and Erler, 2007a, M-284416-01-1, report RA-2511/06; Wolters, 2007a, M-285005-01-1, report RA-2616/06, Mickelson, 1999a, M-240663-01-1, report AA98714495; Fischer and Helfrich, 2007, M-285889-01-1, report RAUBP008; Vaughn and Cosgrove, 2007, M-286216-02-1, report RAUBP011]. Small plots of maize (50–1700 m2) were treated as indicated in Table 54 using knapsack, tractor/ATV mounted or wheelbarrow sprayers with spray volumes ranging from 90–300 L/ha






Field samples of forage (whole plants) were taken at various growth stages: BBCH 13–85 in the EU trials, late milk—dent stage (BBCH 85–87) in the US trials and BBCH 75–85 in the Canadian trials. Forage harvested at growth stage BBCH 85 or later still contains the cobs/kernels, but the material itself will be very dry and is not green anymore. Single field samples were taken in the EU trials, duplicate field samples were taken in the US and Canadian trials. Sample sizes were at least 1 kg (in accordance with FAO manual 2009), except for some DAT = 0 samples (0.1–0.8 kg) as indicated in Table 54. Samples were stored frozen at -18 ºC with a maximum storage period of 327 days (EU trials), 185 days at -10 ºC (AA987144495) or 593 days at -15 ºC (RAUBP008), 189 days at -20 ºC (Canadian trials).

In the EU trials, isoxaflutole and its metabolite IFT-DKN and IFT-BA were determined using HPLC-MS-MS method 00985/M001. This method is considered valid in the range 0.01–0.10 mg/kg for maize forage. Since IFT and IFT-DKN levels in the supervised residue trials on maize forage ranged from < 0.01–13 mg/kg and < 0.01–2.6 mg/kg, respectively, validation at higher levels is desirable. Average concurrent recoveries at 0.01–5.0 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (LOQ) for each analyte. Average concentrations for single field samples are shown in Table 54.

In the 1998 US trials, isoxaflutole and its metabolite IFT-DKN and IFT-BA were determined using HPLC-MS-MS method CAL#019-03 revision 1. The method is considered valid in the range 0.01–0.10 mg/kg for maize forage. Since IFT-BA levels in the supervised trials on maize forage were in the range < 0.01–0.15 mg/kg, validation at higher levels is desirable. Individual concurrent recoveries at 0.01, 0.1 and 10 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 54.

In the 2005 and 2006 Canadian and US trials, isoxaflutole and its metabolites IFT-DKN and IFT-BA were determined using modification C of HPLC-MS-MS method CAL#019-03. This method is considered valid in the range 0.01–0.2 mg/kg IFT, 0.02–0.3 mg/kg IFT-DKN and 0.03–0.3 mg/kg IFT-BA in maize forage. Average concurrent recoveries at 0.01, 0.05 and 0.2 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (reported LOQ). Average

Isoxaflutole

1271

concentrations for duplicate field samples are shown in Table 54. Because of validation results, the LOQs were increased to 0.02 mg/kg for IFT-DKN and 0.03 mg/kg for IFT-BA (indicated by note g).

Note: Storage stability studies on commodities with high water content showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 20 months. Storage stability studies on commodities with high water content showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 12 months but converts to IFT-BA within 23 months of storage. Since maize forage has been stored for longer than 3 months, part of the IFT may have degraded to IFT-DKN. Except for trials in report RAUBP008, maize forage has been stored for less than 12 months and these results are considered reliable. Samples from report RAUBP008 have been stored for longer than 12 months and part of the IFT-DKN may have degraded to IFT-BA. However, in those trials where IFT, IFT-DKN and IFT-BA are all below LOQ, any degradation remains unnoticed and these results are considered reliable. In a few trials where IFT-BA was found to be above LOQ and where the storage period exceeded 12 months, individual results for IFT and IFT-DKN are not considered reliable. These are indicated by note f.

Table 54 Isoxaflutole related residues after pre- or post-emergent treatment in maize forage (whole plant including cobs)

MAIZE FORAGE Location, year, (variety)

FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial (Remarks)


SC a 1 0.10 0.034 13 13 19

0 e 41

2.2 < 0.01

0.71 < 0.01

< 0.01 0.03

2.9 < 0.02

RA-2587/05; R 2005 0623/6


SC b 1 0.099 0.033 13 13 33 85

0 e 40 98

6.2 0.28 < 0.01

1.5 0.06 < 0.01

< 0.01 0.02 0.03

7.7 0.34 < 0.02

RA-2510/06 R 2006 0795/4


SC a 1 0.10 0.034 13 13 35

0 e 41

2.9 < 0.01

1.0 < 0.01

< 0.01 0.01

3.9 < 0.02

RA-2587/05 R 2005 0961/8


SC c 1 c 0.10 0.034 05 18 71 85

41 81 111

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02

RA-2615/06 R 2006 0801/2


SC c 1 c

0.10 0.034 06 18 65 85

45 85 139

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.02 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02

RA-2615/06 R 2006 0802/0

United Kingdom Cambridge (Cambridges

SC a 1 0.10 0.034 13 13 34

0 e 40

3.2 < 0.01

0.53 < 0.01

< 0.01 0.02

3.7 < 0.02

RA-2587/05 R 2005 0959/6

Isoxaflutole

1272


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


hire), 2005, (RK210), Soil: SaL


SC b 1 0.099 0.033 14 14 37 85

0 e 39 106

4.5 0.01 < 0.01

0.42 < 0.01 < 0.01

0.01 0.01 0.03

4.9 0.02 < 0.02

RA-2510/06 R 2006 0796/2


SC c 1 c

0.10 0.034 07 18 71

46 87

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0800/4


SC a 1 0.10 0.034 13 13 19

0 e 40

1.1 < 0.01

0.20 < 0.01

< 0.01 < 0.01

1.3 < 0.02

RA-2587/05 R 2005 0962/6


SC a 1 0.10 0.034 14 14 39

0 e 40

2.7 < 0.01

0.64 < 0.01

< 0.01 0.03

3.3 < 0.02

RA-2587/05 R 2005 0958/8


SC b 1 0.099 0.033 13 13 19 85

0 e 40 110

7.1 < 0.01 < 0.01

1.2 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

8.3 < 0.02 < 0.02

RA-2510/06 R 2006 0073/9

SC c 1 c

0.10 0.034 01 18 69 85

55 94 134

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02

RA-2615/06 R 2006 0627/3


SC c 1 c

0.10 0.034 00 18 63 85

52 91 126

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.03 < 0.01 0.02

< 0.02 < 0.02 < 0.02

RA-2615/06 R 2006 0799/7


SC a

1 0.10 0.034 13 13 34

0 e 40

1.9 < 0.01

0.46 < 0.01

< 0.01 0.01

2.4 < 0.02

RA-2588/05 R 2005 0624/4

S-France Vouille (Poitou-

SC b 1 0.099 0.033 13 13 19 85

0 e 40 116

13 < 0.01 < 0.01

2.6 < 0.01 < 0.01

0.01 < 0.01 < 0.01

16 < 0.02 < 0.02

RA-2511/06 R 2006

Isoxaflutole

1273


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


Charentes) 2006, (dkc 4845) Soil: SiC

0074/7


SC c 1 c

0.10 0.034 06 18 69 85

49 90 115

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.03 0.03 0.03

< 0.02 < 0.02 < 0.02

RA-2616/06 R 2006 0628/1


SC c 1 c

0.10 0.034 01 18 67 85

30 70 109

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02

RA-2616/06 R 2006 0803/9

Spain Vila-sacra (Cataluña) 2005, (DKc6575); Soil: SaL

SC a 1 0.10 0.034 13 13 19

0 e 40

4.9 < 0.01

0.71 < 0.01

0.02 < 0.01

5.6 < 0.02

RA-2588/05 R 2005 0963/4


SC b 1 0.099 0.033 14 14 36 85

0 e 41 77

7.2 < 0.01 < 0.01

1.2 < 0.01 < 0.01

0.01 < 0.01 < 0.01

8.4 < 0.02 < 0.02

RA-2511/06 R 2006 0798/9

SC c 1 c

0.10 0.034 07 18 85

48 99

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2616/06 R 2006 0804/7


SC c 1 c

0.10

0.034 00 18 73 85

32 72 86

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02

RA-2616/06 R 2006 0806/3


SC a 1 0.10 0.034 13 13 35

0 40

3.7 < 0.01

0.95 < 0.01

< 0.01 < 0.01

4.6 < 0.02

RA-2588/05 R 2005 0964/2

Italy San Carlo (FE) Emilia-Romagna, 2006, (PR34N43) Soil: SaC

SC b 1 0.099 0.033 13 13 35 85

0 e 40 88

5.4 < 0.01 < 0.01

0.32 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

5.7 < 0.02 < 0.02

RA-2511/06 R 2006 0797/0

SC c 1 c

0.10 0.034 01 18 63 85

43 84 113

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02

RA-2616/06 R 2006 0805/5

Greece Chalastra

SC a 1 0.10 0.034 13 13 31

0 e 39

4.5 < 0.01

0.34 < 0.01

< 0.01 0.01

4.8 < 0.02

RA-2588/05

Isoxaflutole

1274


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


(Macedonia) 2005, (Decalp 743); Soil: L

R 2005 0965/0


SC a 1 0.10 0.034 13 13 35

0 e 41

3.0 < 0.01

0.61 < 0.01

< 0.01 < 0.01

3.6 < 0.02

RA-2588/05 R 2005 0966/9



De 107 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-01

USA Wilson NC, 1998, (Pioneer 3163) Soil: SaL


De 73 < 0.01 < 0.01 0.15 < 0.02 AA98714495 14495-02



EaDe 109 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-04

USA Percival IA, 2005, (Sucrossco 2009) Soil: SiL

SC a 1 0.13 0.10 12 EaFo ECH

45 76

< 0.01 < 0.01

< 0.02g < 0.02g

< 0.03g

< 0.03g < 0.03g < 0.03g

RAUBP008, UB086-05H

USA Jefferson IA, 2005 (DKC63-81) Soil: L

SC a 1 0.13 0.10 12 ECH 87 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP008, UB091-05H

USA Bagley IA, 2006 (FA6508) Soil: CL

SC a 1 0.13 0.11 12 EaFo 45 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP008, UB091-05HA d



EaDe 88 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-05

USA Shelby MO, 1998, (Novartis NK 6800) Soil: SiL


EaDe 88 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-21

USA Stoddard


LaDo 80 < 0.01 < 0.01 0.014 < 0.02 AA98714495

Isoxaflutole

1275


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


MO, 1998, (Pioneer 3394) Soil: SiL

14495-10


SC a 1 0.13 0.098 12 EaFo Fo

43 104

< 0.01 < 0.01

< 0.02g < 0.02g

< 0.03g 0.034

< 0.03g

< 0.03f,g RAUBP008, UB081-05H


SC a 1 0.13 0.094 12 ECH 79 < 0.01 < 0.02g 0.031 < 0.03f,g RAUBP008, UB088-05H

USA Clarence MO, 2006, (–) Soil: SiL

SC a 1 0.13 0.070 12 EaFo 45 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP008, UB088-05HA d



EaDe 105 < 0.01 < 0.01 0.019 < 0.02 AA98714495 14495-06

USA Fayette OH, 1998, (SC 1068) Soil: SiL


EaDo Do EaDe De De

46 103 110 117 124

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02 < 0.02

AA98714495 14495-19

USA Marysvillle OH, 2005, (Shur Grow SG-751) Soil: CL

SC a 1 0.13 0.079 12 EaFo ECH

45 86

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB089-05H


SC a 1 0.13 0.092 12 EaFo ECH

45 93

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB092-05H



EaDe 95 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-07



EaDe 93 < 0.01 < 0.01 0.015 < 0.02 AA98714495 14495-08

USA Grand Forks ND, 1998, (Mycogen 2110 R3)


EaDe 114 < 0.01 < 0.01 0.014 < 0.02 AA98714495 14495-09

Isoxaflutole

1276


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


Soil: SiL USA Steele ND, 1998, (Mycogen 2110 R3) Soil: SiL


EaDe 108 < 0.01 < 0.01 0.017 < 0.02 AA98714495 14495-16


SC a 1 0.13 0.094 12 EaFo ECH

43 90

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB093-05H



EaDe 91 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-11



LaDo 86 < 0.01 < 0.01 0.012 < 0.02 AA98714495 14495-12



EaDe 91 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-20


SC a 1 0.13 0.10 12 EaFo EaFo EaFo EaFo EaFo Fo

30 37 44 51 58 106

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.02g

< 0.02g < 0.02g

< 0.02g < 0.02g

< 0.02g

< 0.03g < 0.03g < 0.03g < 0.03g < 0.03g < 0.03g

< 0.03g < 0.03g < 0.03g < 0.03g < 0.03g < 0.03g

RAUBP008, UB082-05D


SC a 1 0.13 0.087 12 EaFo Fo

45 88

< 0.01 < 0.01

< 0.02g

< 0.02g 0.034 0.064

< 0.03g < 0.03g

RAUBP008, UB083-05H


SC a 1 0.13 0.080 12 EaFo ECH

45 95

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB094-05H



EaDe 89 < 0.01 < 0.01 0.023 < 0.02 AA98714495 14495-13


LaMi Do EaDe LaDo De

74 81 88 95 102

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02 < 0.02

AA98714495 14495-18

USA WG 1 0.16 0.11 bare EaDe 93 < 0.01 < 0.01 0.036 < 0.02 AA987144

Isoxaflutole

1277


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


Polk NE, 1998, (Northrup King N3030BT) Soil: LSa

soil 95 14495-14

USA Springfield NE, 2005 (NK70T9) Soil: SiL

SC a 1 0.13 0.10 12 EaFo ECH

45 76

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB085-05H

USA Stafford KS, 1998, (Pioneer 3737) Soil: LSa


EaDe 83 < 0.01 < 0.01 0.11 < 0.02 AA98714495 14495-15


SC a 1 0.14 0.10 12 EaFo EaFo EaFo EaFo EaFo EaFo

31 38 45 51 59 94

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.02g

< 0.02g < 0.02g

< 0.02g < 0.02g

< 0.02g

0.030 0.043 0.054 0.046 0.052 0.055

< 0.03f,g < 0.03f,g < 0.03f,g < 0.03f,g < 0.03f,g < 0.03f,g

RAUBP008, UB080-05DA d



EaDe 79 < 0.01 < 0.01 0.012 < 0.02 AA98714495 14495-17


SC a 1 0.13 0.094 12 EaFo ECH

45 67

< 0.01 0.015

< 0.02g

< 0.02g 0.065 0.069

< 0.03 f,g < 0.03f,g

RAUBP008, UB097-05H

USA Bumpass VA, 2005, (Dekalb) Soil: SaCL

SC a 1 0.13 0.098 12 EaFo Fo

45 77

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g

< 0.03g RAUBP008, UB078-05H


SC a 1 0.13 0.078 12 EaFo Fo

45 81

< 0.01 < 0.01

< 0.02g

< 0.02g 0.034 < 0.03g

< 0.03f,g < 0.03g

RAUBP008, UB079-05H


SC a 1 0.13 0.10 12 EaFo ECH

45 100

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB084-05H


SC a 1 0.13 0.076 12 EaFo ECH

43 90

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB095-05H

USA Sabin MN, 2005, (Pioneer

SC a 1 0.14 0.15 12 EaFo ECH

45 118

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03f,g < 0.03g

RAUBP008, UB087-05H

Isoxaflutole

1278


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


39H85) Soil: Si


SC a 1 0.13 0.094 13 EaFo ECH

45 97

< 0.01 < 0.01

< 0.02g

< 0.02g < 0.03g < 0.03g

< 0.03g < 0.03g

RAUBP008, UB090-05H


SC a 1 0.13 0.075 12 EaFo ECH

43 96

< 0.01 < 0.01

< 0.02g

< 0.02g 0.065 0.035

< 0.03f,g < 0.03f,g

RAUBP008, UB096-05H

Canada Clarksburg, ON, 2006, (Pioneer 39F27) Soil: L

SC a 1 0.21 0.11 13 75–85 60 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 01-06H


SC a 1 0.21 0.11 12–13 75–85 61 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 02-06H


SC a 1 0.21 0.11 13 75–85 75–85 75–85 75–85

48 54 61 67

< 0.01 < 0.01 < 0.01 < 0.01

< 0.02g < 0.02g < 0.02g < 0.02g

< 0.03g < 0.03g < 0.03g < 0.03g

< 0.03g < 0.03g < 0.03g < 0.03g

RAUBP011, 03-06D

Canada Breslau, ON, 2006, (Pioneer 39F28) Soil: SaL

SC a 1 0.21 0.11 13 75–85 61 < 0.01 < 0.02g 0.036 < 0.03g RAUBP011, 04-06H

Canada Greenfield, ON, 2006, (Pioneer 39F28) Soil: SaL

SC a 1 0.21 0.11 13 75–85 61 < 0.01 < 0.02g 0.042 < 0.03g RAUBP011, 05-06H


SC a 1 0.22 0.11 13 75–85 60 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 06-06H


SC a 1 0.21 0.11 13 75–85 61 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 07-06H

Canada Paris, ON, 2006 (Dekalb 4057)

SC a 1 0.21 0.11 12–13 75–85 62 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 10-06H

Isoxaflutole

1279


FL no kg ai/ha

kg ai/hL GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq


Soil: SaL Canada Springford, ON, 2006 (DKC42-70) Soil: SaL

SC a 1 0.22 0.11 12–13 75–85 61 < 0.01 < 0.02g 0.061 < 0.03g RAUBP011, 11-06H


SC a 1 0.22 0.11 12–13 75–85 75–85 75–85 75–85

48 53 60 67

< 0.01 < 0.01 < 0.01 < 0.01

< 0.02g < 0.02g < 0.02g < 0.02g

< 0.03g

< 0.03g

0.035 0.034

< 0.03g < 0.03g < 0.03g < 0.03g

RAUBP011, 12-06D


SC a 1 0.23 0.11 13 75–85 61 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 08-06H


SC a 1 0.21 0.10 13 75–85 60 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 09-06H


SC a 1 0.21 0.12 13 75–85 64 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 13-06H

Canada Acton Vale, QC, 2006, (NK N16-N7) Soil: Sa

SC a 1 0.22 0.13 13 75–85 63 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 14-06H

Canada Ste-Madeleine, QC, 2006, (Mycogen 2P172) Soil: SaL

SC a 1 0.21 0.12 13 75–85 61 < 0.01 < 0.02g < 0.03g < 0.03g RAUBP011, 15-06H

Soil: C = Clay, L= loam, Si = silt; Sa= sand; CL = clay loam; CSa = clay sand, CSi= clay silt; LSa = loamy sand; SiC = silty clay; SaCL=sandy clay loam; SiCL = silty clay loam, SiL = silty loam; SaC = sandy clay; SaL = sandy loam, SaSi = sandy silt; GS: EaFo = early forage, Fo = forage, LaMi = late milk; EaDo = early dough, Do = dough; LaDo= late dough; EaDe= early dent; De = dent;; ECH = earliest commercial harvest, 75–85 = kernels in the middle of the cob are yellowish white to dough state. total = sum of IFT and IFT-DKN, expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. a SC formulation contained 20.4% w/w isoxaflutole and 20.3% w/w cyprosulfamide (safener); or 240 g/L each b SC formulation contained 225 g/L isoxaflutole, 150 g/L cyprosulfamide (safener) and 90 g/L thiencarbazone-methyl c A first spray application was performed with an SC formulation (containing 240 g/L isoxaflutole and 240 g/L cyprosulfamide (safener)) at the pre-emergence stage followed by a second application with an SC formulation (containing 225 g/L thiencarbazone-methyl and 225 g/L cyprosulfamide (safener)) at growth stage BBCH 18. Days after last treatment (DAT) are related to the isoxaflutole treatment (i.e. days after the first treatment). d Trials UB088-05HA and UB091-05HA were repeated in 2006 in order to collect early forage samples at DAT = 45. Trial UB080-05DA was repeated in 2006 due to improper application timing in the original trial.

Isoxaflutole

1280

e Sample sizes were less than the required minimum of 1 kg (in accordance with FAO manual 2009): 0.1–0.8 kg at DAT = 0 in report RA-2587/05, RA-2510/06 and RA-2588/05. Samples are considered not representative and trials cannot be selected for STMR or HR derivation, if according to cGAP. f Individual results for IFT and IFT-DKN not considered reliable, since samples were stored for longer than 12 months.

Maize fodder

Supervised trials on maize were conducted in Spain, Italy, Greece, Portugal, Germany, France, Netherlands and the United Kingdom in 2005 and 2006 and in USA and Canada in 1998, 2005 and 2006 [Zimmer and Wieland, 2007, M-282674-01-1, report RA-2587/05; Wolters and Erler, 2007b, M-284423-01-1, report RA-2510/06; Wolters, 2007b, M-285014-01-1, report RA-2615/06; Zimmer, 2006, M-281611-01-1, report RA-2588/05; Wolters and Erler, 2007a, M-284416-01-1, report RA-2511/06; Wolters, 2007a, M-285005-01-1, report RA-2616/06, Mickelson, 1999a, M-240663-01-1, report AA98714495; Fischer and Helfrich, 2007, M-285889-01-1, report RAUBP008; Vaughn and Cosgrove, 2007, M-286216-02-1, report RAUBP011]. Small plots of maize (50-1700 m2) were treated as indicated in table 55 using knapsack, tractor/ATV mounted or wheelbarrow sprayers with spray volumes ranging from 90-300 L/ha






Field samples of maize fodder (plants after removal cobs/kernels) were taken as sweet corn fodder (BBCH 79) or as field corn fodder (mature, BBCH 85–89). Single field samples were taken in the EU trials, duplicate field samples were taken in the USA trials. All sample sizes were at least 1 kg and at least 12 plants (in accordance with FAO manual 2009), except some Canadian trials (0.8–0.9 kg) as indicated in Table 55. All samples were stored frozen with a maximum storage period of 244 days at -18 ºC (EU trials), 185 days at -10 ºC (AA987144495) or 546 days at -15 ºC (RAUBP008), 147days at -20 ºC (Canadian trials).

In the EU trials, isoxaflutole and its metabolite IFT-DKN and IFT-BA were determined using HPLC-MS-MS method 00985/M001. This method is considered valid in the range 0.01–0.01 mg/kg in maize grains and maize forage, but maize fodder has not been validated. Additional validation results for maize fodder are desirable. Concurrent recoveries in maize fodder were not verified, although recoveries for maize grains, sweet corn and maize forage were within 70–120%. Levels in control samples were < 0.01 mg/kg (LOQ) for each analyte. Average concentrations for duplicate field samples are shown in Table 55.

In the 1998 US trials, isoxaflutole and its metabolite IFT-DKN and IFT-BA were determined using HPLC-MS-MS method CAL#019-03 revision 1. This method is considered valid in the range 0.01–0.10 mg/kg for maize fodder. Individual concurrent recoveries at 0.01, 0.1 and 10 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ). Average concentrations for duplicate field samples are shown in Table 55.

In the 2005 and 2006 Canadian and US trials, isoxaflutole and its metabolites IFT-DKN and IFT-BA were determined using modification C of HPLC-MS-MS method CAL#019-03. This modification is considered valid in the range 0.01–0.2 mg/kg for IFT, 0.02–0.3 mg/kg for IFT-DKN and 0.03–0.3 mg/kg for IFT-BA in maize fodder. Average concurrent recoveries at 0.01, 0.05 and 0.2 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (reported LOQ). Average concentrations for duplicate field samples are shown in Table 55. Because

Isoxaflutole

1281

of validation results, the LOQs were increased to 0.02 mg/kg for IFT-DKN and 0.03 mg/kg for IFT-BA (indicated by note h).

Note: Storage stability studies on straw showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 15 months. Storage stability studies on various commodities showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 12 months but converts to IFT-BA within 23 months of storage. Since maize fodder has been stored for longer than 3 months, part of the IFT may have degraded to IFT-DKN. Except for trials in report RAUBP008, maize fodder has been stored for less than 12 months and these results are considered reliable. Samples from report RAUBP008 have been stored for longer than 12 months and part of the IFT-DKN may have degraded to IFT-BA. However, in those trials where IFT, IFT-DKN and IFT-BA are all below LOQ, any degradation remains unnoticed and these results are considered reliable. In a few trials where IFT-BA was found to be above LOQ and where the storage period exceeded 12 months, individual results for IFT and IFT-DKN are not considered reliable. These are indicated by note g.

Table 55 Isoxaflutole related residues after pre- or post-emergent treatment in maize fodder (plant without cobs, or cobs)

MAIZE FODDER Location, year, (variety)

FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial


SC a 1 0.10 0.034 13 79 85 89 cob 89

78 111 127 cob 127

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

0.03 0.03 0.03 < 0.01

< 0.02 < 0.02

< 0.02 < 0.02

RA-2587/05; R 2005 0623/6


SC b 1 0.099 0.033 13 79 77 < 0.01 < 0.01 0.02 < 0.02 RA-2510/06 R 2006 0795/4


SC a 1 0.10 0.034 13 79 85 89 cob 89

90 112 128 cob 128

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

0.04 0.03 0.02 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02

RA-2587/05 R 2005 0961/8


SC c 1 c 0.10 0.034 05 79 91 < 0.01 < 0.01 < 0.01 < 0.02 RA-2615/06 R 2006 0801/2


SC c 1 c 0.10 0.034 06 79 116 < 0.01 < 0.01 0.01 < 0.02 RA-2615/06 R 2006 0802/0


SC a 1 0.10 0.034 13 79 85 89 cob 89

103 124 148 cob 148

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

0.02 0.02 0.02 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02

RA-2587/05 R 2005 0959/6

United Kingdom Cambridge (Cambridgeshire)

SC b 1 0.099 0.033 14 79 93 < 0.01 < 0.01 0.04 < 0.02 RA-2510/06 R 2006 0796/2

Isoxaflutole

1282


FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

2006, (Nexxos); Soil: SaL United Kingdom, EYE (Suffolk) 2006, (Algans); Soil: SiL

SC c 1 c 0.10 0.034 07 79 85

101 124

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2615/06 R 2006 0800/4


SC a 1 0.10 0.034 13 79 85 89 cob 89

116 128 143 cob 143

< 0.01 < 0.01 < 0.01 cob < 0.01

< 0.01 < 0.01 < 0.01 cob < 0.01

< 0.01 < 0.01 < 0.01 cob < 0.01

< 0.02 < 0.02 < 0.02 cob < 0.02

RA-2587/05 R 2005 0962/6


SC a 1 0.10 0.034 14 79 85 89 cob 89

71 83 127 cob 127

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

0.04 0.04 0.04 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02

RA-2587/05 R 2005 0958/8


SC b 1 0.099 0.033 13 79 96 < 0.01 < 0.01 < 0.01 < 0.02 RA-2510/06 R 2006 0073/9

SC c 1 c 0.10 0.034 01 79 120 < 0.01 < 0.01 < 0.01 < 0.02 RA-2615/06 R 2006 0627/3


SC c 1 c 0.10 0.034 00 79 112 < 0.01 < 0.01 < 0.01 < 0.02 RA-2615/06 R 2006 0799/7


SC a

1 0.10 0.034 13 79 85 89 cob 89

83 106 148 cob 148

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

0.03 0.03 0.02 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02

RA-2588/05 R 2005 0624/4

S-France Vouille (Poitou-Charentes) 2006, (dkc 4845) Soil: SiC

SC b 1 0.099 0.033 13 79 98 < 0.01 < 0.01 < 0.01 < 0.02 RA-2511/06 R 2006 0074/7


SC c 1 c 0.10 0.034 06 79 105 < 0.01 < 0.01 0.04 < 0.02 RA-2616/06 R 2006 0628/1


SC c 1 c 0.10 0.034 01 79 91 < 0.01 < 0.01 < 0.01 < 0.02 RA-2616/06 R 2006 0803/9

Spain Vila-sacra (Cataluña)

SC a 1 0.10 0.034 13 79 85

84 98

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

RA-2588/05 R 2005

Isoxaflutole

1283


FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

2005, (DKc6575); Soil: SaL

89 cob 89

141 cob 141

< 0.01 < 0.01

< 0.01 < 0.01

< 0.01 < 0.01

< 0.02 < 0.02

0963/4


SC b 1 0.099 0.033 14 79 67 < 0.01 < 0.01 < 0.01 < 0.02 RA-2511/06 R 2006 0798/9

SC c 1 c 0.10 0.034 07 79 89 < 0.01 < 0.01 < 0.01 < 0.02 RA-2616/06 R 2006 0804/7


SC c 1 c 0.10

0.034 00 79 78 < 0.01 < 0.01 < 0.01 < 0.02 RA-2616/06 R 2006 0806/3


SC a 1 0.10 0.034 13 79 85 89 cob89

79 93 128 cob 128

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02

RA-2588/05 R 2005 0964/2

Italy San Carlo (FE), Emilia-Romagna 2006, (PR34N43) Soil: SaC

SC b 1 0.099 0.033 13 79 75 < 0.01 < 0.01 < 0.01 < 0.02 RA-2511/06 R 2006 0797/0

SC c 1 c 0.10 0.034 01 79 100 < 0.01 < 0.01 < 0.01 < 0.02 RA-2616/06 R 2006 0805/5


SC a 1 0.10 0.034 13 79 85 89 cob 89

77 105 137 cob 137

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02

RA-2588/05 R 2005 0965/0


SC a 1 0.10 0.034 13 79 85 89 cob 89

80 100 133 cob 133

< 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01

0.01 0.02 0.03 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02

RA-2588/05 R 2005 0966/9



Mat 143 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-01

USA Wilson NC, 1998, (Pioneer 3163) Soil: SaL


Mat 133 < 0.01 < 0.01 0.023 < 0.02 AA98714495 14495-02



Mat 139 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-04

USA Percival IA, 2005,

SC a 1 0.13 0.10 12 ECH 125 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP008, UB086-05H

Isoxaflutole

1284


FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

(Sucrossco 2009) Soil: SiL USA Jefferson IA, 2005 (DKC63-81) Soil: L




Mat 132 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-05

Soil: SiL WG 1 0.16 0.090 bare soil

Mat 140 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-21

USA Stoddard MO, 1998, (Pioneer 3394) Soil: SiL


Mat 115 < 0.01 < 0.01 0.013 < 0.02 AA98714495 14495-10




SC a 1 0.13 0.094 12 ECH 102 < 0.01 < 0.02h 0.035 < 0.03g,h RAUBP008, UB088-05H



Mat 153 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-06

(SC 1068) Soil: SiL


Dent Dent Mat Mat Mat

124 131 138 145 152

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 0.013 < 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02 < 0.02

AA98714495 14495-19

USA Marysvillle OH, 2005, (Shur Grow SG-751) Soil: CL






Mat 143 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-07



Mat 144 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-08

USA Grand Forks


Mat 146 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495

Isoxaflutole

1285


FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

ND, 1998, (Mycogen 2110 R3) Soil: SiL

14495-09

USA Steele ND, 1998, (Mycogen 2110 R3) Soil: SiL


Mat 138 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-16





Mat 142 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-11



Mat 111 < 0.01 < 0.01 0.022 < 0.02 AA98714495 14495-12



R6 134 < 0.01 < 0.01 < 0.01 < 0.02 AA98714495 14495-20


SC a 1 0.13 0.10 12 ECH 129 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP008, UB082-05D







Mat 106 < 0.01 < 0.01 0.047 < 0.02 AA98714495 14495-13


Dent MatMat Mat Mat

103 109 116 123 130

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.01 < 0.01 < 0.01 < 0.01 < 0.01

< 0.02 < 0.02 < 0.02 < 0.02 < 0.02

AA98714495 14495-18

USA Polk NE, 1998, (Northrup King N3030BT) Soil: LSa


Mat 109 < 0.01 < 0.01 0.071 < 0.02 AA98714495 14495-14

USA Springfield


Isoxaflutole

1286


FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

NE, 2005 (NK70T9) Soil: SiL USA Stafford KS, 1998, (Pioneer 3737) Soil: LSa


Mat 103 < 0.01 < 0.01 0.089 < 0.02 AA98714495 14495-15


SC a 1 0.14 0.10 12 ECH 119 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP008, UB080-05DA d



Mat 104 < 0.01 < 0.01 0.022 < 0.02 AA98714495 14495-17



USA Bumpass VA, 2005, (Dekalb) Soil: SaCL








USA Sabin MN, 2005, (Pioneer 39H85) Soil: Si

SC a 1 0.14 0.15 12 ECH 134 e

< 0.01 < 0.02h < 0.03h < 0.03h RAUBP008, UB087-05H





Canada SC a 1 0.21 0.11 13 – 114 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011,

Isoxaflutole

1287


FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

Clarksburg, ON, 2006, (Pioneer 39F27) Soil: L

01-06H


SC a 1 0.21 0.11 12-13 – 112 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 02-06H


SC a 1 0.21 0.11 13 – 96 103 110 117

< 0.01 < 0.01 < 0.01 < 0.01

< 0.02h < 0.02h

< 0.02h < 0.02h

< 0.03h < 0.03h

< 0.03h < 0.03h

< 0.03h < 0.03h

< 0.03h < 0.03h

RAUBP011, 03-06D

Canada Breslau, ON, 2006, (Pioneer 39F28) Soil: SaL

SC a 1 0.21 0.11 13 – 110 < 0.01 < 0.02h 0.032 < 0.03h RAUBP011, 04-06H

Canada Greenfield, ON, 2006, (Pioneer 39F28) Soil: SaL

SC a 1 0.21 0.11 13 – 110 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 05-06H f


SC a 1 0.22 0.11 13 110 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 06-06H


SC a 1 0.21 0.11 13 – 113 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 07-06H

Canada Paris, ON, 2006 (Dekalb 4057) Soil: SaL

SC a 1 0.21 0.11 12-13 – 108 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 10-06H

Canada Springford, ON, 2006 (DKC42-70) Soil: SaL

SC a 1 0.22 0.11 12-13 – 110 < 0.01 < 0.02h 0.13 < 0.03h RAUBP011, 11-06H


SC a 1 0.22 0.11 12-13 – 96 103 110 117

< 0.01 < 0.01 < 0.01 < 0.01

< 0.02h < 0.02h < 0.02h

< 0.02h

0.16 0.087 0.070 0.050

< 0.03h < 0.03h < 0.03h

< 0.03h

RAUBP011, 12-06D


SC a 1 0.23 0.11 13 – 111 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 08-06H


SC a 1 0.21 0.10 13 – 111 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 09-06H f


SC a 1 0.21 0.12 13 – 111 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011, 13-06H

Canada SC a 1 0.22 0.13 13 – 110 < 0.01 < 0.02h < 0.03h < 0.03h RAUBP011,

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FL no kg ai/ha

kg ai/hL

GS appl

GS harv

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

total mg/kg eq

Study Trial

Acton Vale, QC, 2006, (NK N16-N7) Soil: Sa

14-06H

Canada Ste-Madeleine, QC, 2006, (Mycogen 2P172) Soil: SaL

SC a 1 0.21 0.12 13 – 111 < 0.01 < 0.02h 0.080 < 0.03h RAUBP011, 15-06H

Soil: C = Clay, L= loam, Si = silt; Sa= sand; CL = clay loam; CSa = clay sand, CSi= clay silt; LSa = loamy sand; SiC = silty clay; SaCL=sandy clay loam; SiCL = silty clay loam, SiL = silty loam; SaC = sandy clay; SaL = sandy loam, SaSi = sandy silt; GS mat = mature; R6 = reproductive stage 6, equivalent to BBCH 89, ECH = earliest commercial harvest total = sum of IFT and IFT-DKN, expressed as isoxaflutole equivalents. Since the molecular weight of IFT-DKN is identical to the molecular weight of the parent compound (MW 359.3), no correction for molecular weight is necessary. a SC formulation contained 20.4% w/w isoxaflutole and 20.3% w/w cyprosulfamide (safener); or 240 g/L each b SC formulation contained 225 g/L isoxaflutole, 150 g/L cyprosulfamide (safener) and 90 g/L thiencarbazone-methyl c A first spray application was performed with an SC formulation (containing 240 g/L isoxaflutole and 240 g/L cyprosulfamide (safener)) at the pre-emergence stage followed by a second application with an SC formulation (containing 225 g/L thiencarbazone-methyl and 225 g/L cyprosulfamide (safener)) at growth stage BBCH 18. Days after last treatment (DAT) are related to the isoxaflutole treatment (i.e. days after the first treatment). d Trial UB080-05D was repeated in 2006 as UB080-05DA due to improper application timing in the original trial. e Trial UB087-05H was harvested at DAT = 133 for grain and DAT = 134 for straw. Since grain and straw are derived from the same commodity it is assumed that the straw has been left to dry for 1 day in the field before sampling. f Sample sizes were less than the required minimum of 1 kg (in accordance with FAO manual 2009): 0.8 kg. Samples are considered not representative and trials cannot be selected for MRL derivation, if according to cGAP. g Individual results for IFT and IFT-DKN are not considered reliable because samples were stored for longer than 12 months. h Results reported below 0.02 mg/kg IFT-DKN and below 0.03 mg/kg IFT-BA, were reported as < 0.02 mg/kg IFT-DKN and < 0.03 mg/kg IFT-BA because of validation results.

Miscellaneous forage and fodder crops

Supervised trials were available for sugarcane forage.

Sugar cane forage

Supervised trials on sugarcane were conducted in Australia during the 1996–1998 growing seasons [Davis and Keats, 1999a, M-284383-01-1, Report AK99009; Davis and Keats, 1999b, M-284347-01-1, Report AK99010; Davis and Keats, 1999c, M-284356-01-1, Report AK99011; Davis and Keats, 1999d, M-284369-01-1, Report AK99012; Davis and Keats, 1999f, M-284324-01-1, Report AK99013; Davis and Keats, 1999e, M-284408-01-1, Report AK99017], in Mexico during the 1999 growing season [Gough, 2000, M-238729-01-1, Study 99717930] and in Brazil during the 1998–1999 growing season [Tornisielo, 2000b, M-287041-01-1, report USP BRA98R27-1; Tornisielo, 2000a, M-287038-01-1 , report USP BRA98R27-2].

In Australia, small plots of sugar cane (15–40 m2) were treated as indicated in Table 56 using plot boom sprayers or spray bars with spray volumes ranging from 238–265 L/ha. In two parallel plots sugarcanes in the 5–8 leaf stage received a single soil application of isoxaflutole at two different application rates.

Single field samples of whole canes were collected at a harvestable maturity at DAT = 133–209. Samples from each plot were taken from at least 12 separate plants. The top 30 cm of the whole canes were then cut off using secateurs to provide forage (tops). At least 1 kg of sample was supplied from each plot (in accordance with the FAO 2009 manual). All samples were stored frozen for a

Isoxaflutole

1289

maximum storage period of 5 months at -20 ºC (AK 99009, AK 99010), 10 months at -20 ºC (AK 99011, AK 99012) or 19–20 months at -20 ºC (AK 99013, AK 99017).

Samples were analysed using GC-MS-MS modification A of method 00473 (common moiety method), where IFT, IFT-DKN and IFT-BA are converted into RPA 204497 and total residues are expressed as isoxaflutole equivalents. This method is considered valid at 0.01 mg/kg in sugarcane forage. Average concurrent recoveries at 0.01 mg/kg were within 70–120% for each analyte. Levels in control samples were < 0.01 mg/kg (LOQ). Average results of the replicate field samples are reported in Table 56.

Note: Storage stability studies on commodities with high water content showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 20 months. Since sugarcane forage has been stored for 20 months or shorter, all results are considered reliable.

Table 56 Isoxaflutole related residues after pre-harvest soil directed treatment in sugarcane (forage/tops)

SUGAR CANE FORAGE Location, year, (variety)

FL No kg ai/ha

kg ai/hL

GS appl GS harv

DAT

IFT mg/kg

RPA 202 248 mg/kg

IFT-BA mg/kg

Total mg/kg eq

Study, Trial

Australia Tolga Mareeba, QLD, 1998, (Q 120) Soil: SaCL

WG 1 0.150 0.057 120 cm CH 166 NA NA NA < 0.01 AK99009

WG 1 0.225 0.085 120 cm CH 166 NA NA NA < 0.01 Australia Atherton, QLD, 1998, (Q 124) Soil: CL

WG 1 0.150 0.057 120 cm CH 133 NA NA NA < 0.01 AK99010

WG 1 0.225 0.0856 120 cm CH 133 NA NA NA < 0.01 Australia Murwillumbah QLD, 1997/98, (Q 170) Soil: peat

WG 1 0.150 0.058 110 cm, (30 cm TVD)

CH 167 NA NA NA < 0.01 AK99011

WG 1 0.225 0.086 110 cm, (30 cm TVD)

CH 167 NA NA NA < 0.01

Australia Rocky Point, QLD, 1997/98 (Q 154) Soil: peat

WG 1 0.150 0.058 100 cm (30 cm TVD)

CH 193 NA NA NA < 0.01 AK99012

WG 1 0.225 0.086 100 cm (30 cm TVD)

CH 193 NA NA NA < 0.01

Australia Bundaberg, QLD, 1997 (Q 124) Soil: podzolic

WG 1 0.150 0.059 60 cm TVD

CH 209 NA NA NA < 0.01 AK99013

WG 1 0.300 0.12 60 cm TVD

CH 209 NA NA NA < 0.01

Australia WG 1 0.150 0.063 5–7 CH 206 NA NA NA < 0.01 AK99017

Isoxaflutole

1290

SUGAR CANE FORAGE Location, year, (variety)

FL No kg ai/ha

kg ai/hL

GS appl GS harv

DAT

IFT mg/kg

RPA 202 248 mg/kg

IFT-BA mg/kg

Total mg/kg eq

Study, Trial

Jardine QLD, 1996 (Q 96) Soil: baratter cracking clay

leaves (25–30 cm TVD)

WG 1 0.300 0.126 5–7 leaves (25–30 cm TVD)

CH 206 NA NA NA < 0.01

Soil: C = clay, CL = clay loam, SaCL = sandy clay loam Total = Total residues of IFT = measured sum of IFT, RPA 20248 and IFT-BA residues, expressed as isoxaflutole equivalents. The total residue is determined via a common moiety method which converts IFT, IFT-DKN and IFT-BA into the derivative RPA 204497 TVD = top visible dewlap CH = commercial harvest (normal crop maturity, marketable specimens)

FATE OF RESIDUES IN STORAGE AND PROCESSING

In storage

No data on the fate of isoxaflutole-derived residues during storage under warehouse conditions are available.

In processing

The Meeting received processing studies on soya beans, maize and sugarcane. The effect of processing on the nature of residues was not investigated since isoxaflutole related residues are generally very low.

The processing factors (residue processed commodity divided by residue RAC) were calculated based on the total isoxaflutole-derived residues (sum of IFT and IFT-DKN, expressed as isoxaflutole). For the calculation of the total isoxaflutole-derived residues any residues < LOQ were considered to be equal to the LOQ. When the total isoxaflutole-derived residues in the raw agricultural commodity were < LOQ no processing factor was derived. When the total isoxaflutole-derived residues in the processed commodity were < LOQ the calculated processing factor is reported with a "less than" (<) symbol.

Soya bean processing

A field trial was conducted in the US in 2009 [Fischer, 2010, M-368662-01-1] to measure the magnitude of isoxaflutole residues in soya bean processed commodities. An SC formulation was applied on glyphosate/HPPD resistant soya bean at an exaggerated rate of 0.525 kg ai/ha. Application spray volumes were 122–124 L/ha and Induce (0.5% v/v), AMS (2% w/w), Interlock (6 oz/A = 0.44 L/ha), and Defoamer (4 oz/100 gal = 31 mL/hL) were used as adjuvants in the tank mix. Applications were made using ground-based equipment. A bulk sample of soya bean seed was collected at DAT = 86 at the normal growth stage for commercial harvest (BBCH 89) from 12 different areas of the plot. Soya bean processing was performed using procedures that simulated commercial practices.

Isoxaflutole

1291

Aspirated grain fractions

The bulk soya bean seed sample (1463.2 lbs = 664 kg) was dried at 110–135 °F (i.e. 43–57 ºC) in an oven until the moisture was between 10.0 and 13.0%. The moisture adjusted sample (1400 lbs = 635 kg) was split into two batches for dust generation. Each subsample was placed in a dust generation room containing a holding bin, two bucket conveyors, and a screw conveyor. As the sample travelled in the system for 120 minutes, aspiration removed the light impurities in the sample (grain dust, 0.7 lbs = 0.318 kg). Collected grain dust was classified on a sieve shaker equipped with the following sieves: 2360 micron (8 mesh); 2000 micron (10 mesh); 1180 micron (16 mesh); 850 micron (20 mesh); and 425 micron (40 mesh). After classification, the material through the 2360 micron sieve was recombined to produce the aspirated grain fractions (AGF, 0.172 kg).

Cleaned whole soya bean processing

Following generation of aspirated grain fractions, a representative sample of aspirated soya beans (200.0 lbs = 90.7 kg) was removed and cleaned further by aspiration and screening. Light impurities were separated from the sample using an aspirator. After aspiration, the sample was screened using a cleaner to separate large and small foreign particles (screenings) from the whole soya bean sample. Resulting fractions were cleaned whole soya beans (183.6 lbs = 83.3 kg), light impurities (0.7 lbs = 0.318 kg), small screenings (2.9 lbs = 1.32 kg) and large screenings (12.8 lbs = 5.81 kg).

Soya bean oil processing

A portion of the cleaned whole soya beans (70.0 lbs = 31.8 kg) was fed into a roller mill to crack the hull and liberate the kernel. After hulling, the material was passed through an aspirator to separate soya bean hulls and kernels. Resulting fraction were soya bean hulls (5.9 lbs = 2.68 kg) and soya bean kernels (64.1 lbs = 29.1 kg).

A portion of the soya bean kernels (30.0 lbs = 13.6 kg) was taken for further processing. The moisture content of the kernel material was determined and adjusted up to 13.5%. Moisture adjusted kernel was allowed to temper for a minimum of twelve hours prior to further processing. Moisture adjusted kernel material was heated to 160–175 °F (i.e. 71–79 ºC) in a mixer and flaked in a flaking roll with a gap setting of 0.008–0.013 " (i.e. 0.20–0.33 mm). Flakes were extruded in a continuous processor, where they were turned into collets by direct steam injection and compression. Collets exited the processor at 200–250 °F (i.e. 93–121 ºC). After extrusion, the collets were dried in the oven at 150–180 °F (i.e. 66–82 ºC) for 30–40 minutes. Collets were placed in separate stainless steel batch extractors and submerged in 120–140 °F (i.e. 49–60 ºC) hexane (solvent). After 30 minutes, the miscella (crude oil and hexane) was drained and fresh hexane was added to repeat the cycle two more times. Final two washes were for 15 minutes each. Extracted collets were desolventized in a paddle mixer by heating to 210–220 °F (i.e. 99–104 ºC) and removing from the mixer. Resulting fractions were miscella (2.815 kg) and soya bean meal (21.4 lbs = 9.71 kg).

A portion of the miscella (2.0 kg) was passed through a laboratory vacuum evaporator unit to separate the crude oil and hexane. Crude oil was heated to 195–205 °F (i.e. 91–96 ºC) for hexane removal and filtered. Percent free fatty acid (FFA) was determined for the crude oil. Based on the FFA, a weighed amount of crude oil and 14° Baume NaOH was placed in a water bath at 68–75 °F (i.e. 20–24 ºC) and mixed for 90 minutes at high RPM, and then for 20 minutes at low RPM and 145–153 °F (i.e. 63–67 ºC). Neutralized oil was then centrifuged. Refined oil was decanted and filtered. Resulting fractions were: alkali refined oil (1.85 kg) and soapstock. Soapstock was discarded.

A portion of the alkali refined oil (1.2 kg) was heated to 104–122 °F (i.e. 40–50 ºC), activated bleaching earth added (1.0% by weight of oil), and placed under vacuum. Temperature was increased to 185–212 °F (i.e. 85–100 ºC), and held for 10 to 15 minutes. After reducing the temperature, the bleached oil was filtered. Resulting fractions were bleached oil (1.149 kg) and spent bleaching earth.

A portion of the bleached oil (1.0 kg) was steam bathed for 28–32 minutes under vacuum and temperature held between 428–446 °F (i.e. 220–230 ºC). During the cooling period a 0.5% citric acid

Isoxaflutole

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solution was added (1 mL per 100 grams of oil deodorized). Resulting fractions was deodorised oil (RBD oil, 0.986 kg) and deodoriser distillates.

Soymilk processing

A portion of the cleaned whole soya beans (0.70 kg) was washed and soaked in water (3.5 kg) for at least 12 hours. A portion of the soaked beans (1.67 kg) were ground and filtered to remove solids and the soymilk was cooked at 195–205 °F (i.e. 91–96 ºC) for 9–11 minutes. Resulting fractions were soymilk (2.873 kg) and okara (soy pulp) (0.703 kg).

Samples of soya bean seed (RAC) were stored frozen (below 0 ºC) with a maximum storage period of 153 days (5 months) until analysis. Samples of soya bean seed (RAC) were stored frozen (below 0 ºC) for 193 days before being processed. Samples of processed commodities were stored frozen at -15 ºC or lower with a maximum storage period of 11 days.

The soya bean seed (RAC) samples and resultant soya bean processed commodities (soya bean meal, soya bean hulls, soya bean oil, soymilk, and soya bean aspirated grain fractions) were analysed in triplicate to determine isoxaflutole and IFT-DKN by modification A of HPLC-MS-MS method IS-004-P10-01. This method is considered valid in the range 0.01–0.1 mg/kg in dry soya bean seed, soya bean meal, soya bean hulls, soya bean oil, soymilk and 0.01–0.5 mg/kg in soya bean aspirated grain fractions. Average concurrent recoveries for mixtures of IFT and IFT-DKN each at 0.01–0.1 mg/kg for soya bean seed, soya bean meal, soya bean hulls, soya bean oil, soymilk or 0.01–0.5 mg/kg for soya bean aspirated grain fractions were within 70–120% for each analyte. Levels in control samples were < 0.003 mg/kg (0.3 LOQ) for each analyte. Average concentrations of IFT, IFT-DKN (n=3 for replicate analytical portions) and corresponding processing factors are indicated in Table 57. The percent dry matter determination on soya bean seed, meal, hulls, and aspirated grain fractions was 89%, 96%, 89%, and 90%, respectively.

Note: Storage stability studies on various commodities showed that total residues (sum of IFT, IFT-DKN and IFT-BA) were stable for at least 11–20 months. Storage stability studies on various commodities showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage and IFT-DKN is stable for at least 16 months in commodities with high oil content and 6 months in all other commodities but converts to IFT-BA within 23 months of storage. Since processed soya bean commodities have been stored for less than 3 months, individual results of IFT and IFT-DKN are considered reliable. Since dry soya beans have been stored for longer than 6 months, part of the IFT may have degraded to IFT-DKN. Since dry soya beans have been stored for less than 16 months, levels of IFT-DKN are not further degraded to IFT-BA and can be considered reliable. If the residue definition includes both IFT and IFT-DKN, processing factors are considered reliable.

Table 57 Processing data on soya beans

Location, Year, (Variety)

Application details

Portion analysed

DAT IFT mg/kg

IFT-DKN mg/kg

IFT-BA mg/kg

Total mg/kg

PF Study Trial No

Springfield USA, 2009 (Stine FG72)

1× 0.525 kg ai/ha at BBCH 60 SC formulation broadcast foliar spray

soya bean seed 86 < 0.01 0.042 NA 0.052 – RAISP005 IS001-09PA

soya bean meal 86 < 0.01 0.051 NA 0.061 1.2 soya bean hulls 86 < 0.01 0.031 NA 0.041 0.79 soya bean RBD oil

86 < 0.01 < 0.01 NA < 0.02 < 0.4

soy milk 86 < 0.01 < 0.01 NA < 0.02 < 0.4 soya bean aspirated grain fractions

86 0.066 0.246 NA 0.312 6.0

PF is based on total residue (sum of IFT and IFT-DKN and expressed as IFT)

Maize processing

A processing study on maize using wet and dry milling was performed [Mickelson, 1999b, M-240664-01-1]. Since the residues in the raw agricultural commodity (maize grains) and the processed

Isoxaflutole

1293

commodities (grits, flour, meal, starch, refined) were below the LOQ (0.01 mg/kg) in all samples, processing factors could not be derived and therefore the study was not summarized.

Sugarcane processing

Six processing studies on sugarcane were performed to get juice and bagasse [Davis and Keats, 1999a/b/c/d/e/f, M-284383-01-1, M-284347-01-1, M-284356-01-1, M-284369-01-1, M-284408-01-1, M-284324-01-1]. Since the residues in the raw agricultural commodity (raw canes) and the processed commodities (juice, bagasse) were < LOQ (0.01 mg/kg) in all samples, processing factors could not be derived and therefore the study was not summarized.

Residues in the edible portion of food commodities

No data submitted.

RESIDUES IN ANIMAL COMMODITIES

Direct animal treatments

Not relevant for the present use pattern.

Farm animal feeding studies

The Meeting received information on feeding studies in dairy cows and laying hens.

Study 1

A cow feeding study was conducted in the USA in 1995 in order to determine the transfer of isoxaflutole related residues from cattle feed into milk, muscle, fat, liver and kidney [Tew, 1995a, M-192311-01-1]. The study was conducted with 14 Holstein lactating cows of 1058–1709 lbs = 480–775 kg body weight (3 to 6-years old), which were distributed in four groups. Group I was the control group and contained two animals. Groups II, III and IV contained four cows per group. The cows were orally dosed once a day in the morning following milking. Cows in group II, III and IV were dosed for 42 days using gelatine capsules fortified with the appropriate amount of isoxaflutole. Group I animals received a placebo containing cellulose powder. Individual daily doses were calculated and adjusted on a weekly basis, based on the group average feed intake from the previous week (16.1–20.2 kg dry matter per cow). Animals in Group II, III and IV were treated with isoxaflutole at actual dose levels of 4.7, 14.4, and 45.5 mg/kg dw feed, respectively, corresponding to 1×, 3×, and 10× dose groups. Equivalent mg/kg bw concentrations were not stated. Cows were milked twice each day. Pooled milk samples for analysis were collected on days 0 (24 hr before first dosing), 1, 4, 8, 11, 15, 18, 22, 25, 27, 31, 33, 36, 39 and 41. For each of these study days, the PM milk samples were refrigerated overnight and combined with the following morning's AM milk sample to get a pooled (500 g) sample in proportion with AM and PM milk production. Test animals were slaughtered within 7.5 hours after receiving their final dose. Samples of muscle (2 kg composite of thigh and loin), fat (1 kg composite of perirenal and omental), liver (1 kg composite from distal portion of each lobe) and kidney (both) were taken from each animal. The samples were stored at –15 ºC or lower for 23–66 days for milk and 83–88 days for tissues.

Single milk samples were analysed for IFT, IFT-DKN, RPA 205834 and IFT-BA using HPLC-UV method RPAC #44882 (= RPA #45532 milk part). The method is considered valid in the range 0.02–2.0 mg/kg IFT, IFT-DKN, RPA 205834 and IFT-BA in milk. Average concurrent recoveries (at 0.02, 0.04 or 0.1 mg/kg) ranged between 70–120% for IFT, IFT-DKN, RPA 205834 and IFT-BA. Residue levels in control samples were either not detected or < LOQ.

Single homogenised tissue samples were analysed for IFT, IFT-DKN, RPA 205834 and RPA 207048 using an HPLC-MS-MS method RPAC #44996, version 1.0. The recovery of residues was only tested at 0.25 mg/kg level. Isoxaflutole is partially degraded during extraction to IFT-DKN and

Isoxaflutole

1294

cannot be determined on its own. Average concurrent recoveries for muscle and fat (at 0.05 mg/kg) ranged between 70–120% for each analyte. Average concurrent recoveries for kidney (at 0.05, 0.45, 0.60 mg/kg) and liver (at 0.05, 0.25, and 2.0 mg/kg) ranged between 70–120% for each analyte. It should be noted that separate recovery samples were prepared and analysed: one for IFT and one for the other compounds. Isoxaflutole is unstable and rapidly metabolizes to IFT-DKN. Therefore, IFT recovery was determined by spiking a control sample with IFT only and the recovery was based on the sum of IFT and IFT-DKN from the analysis. Residue levels in control samples were either not detected or < LOQ.

For Group III, residues in milk of IFT, IFT-DKN, RPA 205834 and IFT-BA were always found to be < 0.02 mg/kg each (Day 0, 4, 8, 22, 36, 39 and 41 analysed). Therefore, milk from Group II was not analysed. For Group IV, residues in milk are summarized in Table 58. In all milk samplings from Group IV (45.5 mg/kg in feed), IFT and IFT-BA were always < 0.02 mg/kg. No plateau was reached and therefore all residues from day 1 and onwards were taken into account for average levels in milk. The levels of isoxaflutole and its metabolites in tissues of cows dosed for 42 consecutive days are summarized in Table 59. None of the results presented in these tables have been corrected for recoveries.

Note: Storage stability studies showed that storage periods of 23–66 days for milk and 83–88 days for tissues will not result in breakdown of IFT, IFT-DKN, or RPA 205834. The results for these compounds are considered reliable. However, stability for RPA 207048 is not guaranteed and this compound may have degraded to about 50% of its original level in tissues. Levels of RPA 207048 in bovine liver at or above the LOQ were therefore multiplied by a factor of 2 to correct for possible degradation.

Note: Milk samples were not analysed for RPA 207048, although this metabolite was found at levels of 0.009 mg/kg eq in a 10 ppm metabolism study in goat (15.0% TRR as RPA 207048 and 75% TRR as IFT-DKN + RPA 205834+RPA 207048). To get total residues B (sum of IFT, IFT-DKN, RPA 205834 and RPA 207048), the sum of IFT, IFT-DKN and RPA 205834 was multiplied by a factor 1.25 to correct for this omission.

Conclusion: The cow feeding study conducted with isoxaflutole shows that residues were mainly transferred to kidney and liver and consisted of metabolites IFT-DKN and RPA 205834. In liver and kidney, there was a linear relationship between the level of isoxaflutole administered to the test animals and the resulting total residue levels.

Table 58 Isoxaflutole related residues in milk for Group IV (45.5 mg/kg in feed)

Sampling Cow IFT mg/kg

IFT-DKN mg/kg

RPA 205834 mg/kg

IFT-BA mg/kg

Total A mg/kg

Total B mg/kg

Day 0 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 < 0.04 < 0.04 < 0.04

< 0.075 < 0.075 < 0.075 < 0.075

Day 1 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 < 0.04 < 0.04 < 0.04

< 0.075 < 0.075 < 0.075 < 0.075

Day 4 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

0.020 0.030 0.023 0.027

< 0.02 < 0.02 < 0.02 < 0.02

NA 0.040 0.050 0.043 0.047

0.075 0.087 0.079 0.084

Day 8 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 0.020 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 < 0.04 0.040 < 0.04

< 0.075 < 0.075 0.075 < 0.075

Day 11 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 0.020 0.033 0.022

< 0.02 < 0.02 0.028 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 0.040 0.053 0.042

< 0.075 0.075 0.10 0.077

Day 15 892 897

< 0.02 < 0.02

< 0.02 0.022

< 0.02 < 0.02

< 0.02 < 0.02

< 0.04 0.042

< 0.075 0.077

Isoxaflutole

1295

Sampling Cow IFT mg/kg

IFT-DKN mg/kg

RPA 205834 mg/kg

IFT-BA mg/kg

Total A mg/kg

Total B mg/kg

900 902

< 0.02 < 0.02

0.031 0.023

< 0.02 < 0.02

< 0.02 < 0.02

0.051 0.043

0.089 0.079

Day 18 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 < 0.04 < 0.04 < 0.04

< 0.075 < 0.075 < 0.075 < 0.075

Day 22 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 0.034 0.028 0.023

< 0.02 < 0.02 0.025 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 0.054 0.048 0.043

< 0.075 0.092 0.091 0.079

Day 25 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

0.024 0.035 0.036 0.023

< 0.02 < 0.02 0.027 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

0.044 0.055 0.056 0.043

0.080 0.094 0.10 0.079

Day 27 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 0.032 0.024 0.023

< 0.02 < 0.02 0.026 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 0.052 0.044 0.043

< 0.075 0.090 0.087 0.079

Day 31 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 < 0.02 0.023 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 < 0.04 < 0.04 < 0.04

< 0.075 < 0.075 0.079 < 0.075

Day 33 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

0.020 0.030 0.024 0.028

< 0.02 < 0.02 0.029 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

0.040 0.050 0.044 0.048

0.075 0.087 0.091 0.085

Day 36 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 0.027 0.022 0.020

< 0.02 < 0.02 0.028 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 0.047 0.042 0.040

< 0.075 0.084 0.087 0.075

Day 39 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

0.024 0.026 0.025 0.024

< 0.02 < 0.02 0.021 0.022

< 0.02 < 0.02 < 0.02 < 0.02

0.044 0.046 0.045 0.044

0.080 0.082 0.082 0.082

Day 41 892 897 900 902

< 0.02 < 0.02 < 0.02 < 0.02

< 0.02 0.023 < 0.02 < 0.02

< 0.02 < 0.02 0.022 < 0.02

< 0.02 < 0.02 < 0.02 < 0.02

< 0.04 0.043 < 0.04 < 0.04

0.075 0.079 0.077 0.075

Average day 1–41

– < 0.02 0.023 0.021 < 0.02 0.043 0.080

Highest residue

900-day 25 < 0.02 0.036 0.027 < 0.02 0.056 0.10

Total A = sum of IFT and IFT-DKN, expressed as isoxaflutole. Conversion factor of IFT-DKN to isoxaflutole equivalents is 1.00. Residues below LOQ were calculated as being at the LOQ = 0.02 mg/kg. Total B = sum of IFT, IFT-DKN, RPA 205834 and RPA 207048, expressed as isoxaflutole. Conversion factor of IFT-DKN to isoxaflutole equivalents is 1.00; conversion factor for RPA 205834 to isoxaflutole equivalents is 359.3/361.3 = 0.994. Residues below LOQ were calculated as being at the LOQ = 0.02 mg/kg. Milk was not analysed for RPA 207048, although this metabolite was found at levels of 0.009 mg/kg eq in a 10 ppm metabolism study in goat (15.0% TRR as RPA 207048 and 75% TRR as IFT-DKN + RPA 205834+RPA 207048). The sum of IFT, IFT-DKN and RPA 205834was therefore multiplied by a factor 1.25 to correct for this omission.

Table 59 Isoxaflutole related residues in tissues of cows

Matrix Analyte Group II 4.7 mg/kg in feed (dry matter)

Group III 14.4 mg/kg in feed (dry matter)

Group IV 45.5 mg/kg in feed (dry matter)

Animal I.D. mg/kg Animal I.D. mg/kg Animal I.D. mg/kg Muscle IFT NA NA 892

897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

IFT- NA NA 892 < 0.05

Isoxaflutole

1296




Animal I.D. mg/kg Animal I.D. mg/kg Animal I.D. mg/kg DKN 897

900 902

< 0.05 < 0.05 < 0.05

RPA 205834

NA NA 892 897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

RPA 207048

NA NA 892 897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

Total A NA NA 892 897 900 902 average

< 0.1 < 0.1 < 0.1 < 0.1 < 0.1

Total B NA NA 892 897 900 902 average

< 0.2 < 0.2 < 0.2 < 0.2 < 0.2

Fat IFT NA NA 892 897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

IFT-DKN

NA NA 892 897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

RPA 205834

NA NA 892 897 900 902

< 0.05 0.064 0.090 0.065

RPA 207048

NA NA 892 897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

Total A NA NA 892 897 900 902 average

< 0.1 < 0.1 < 0.1 < 0.1 < 0.1

Total B NA NA 892 897 900 902 average

< 0.2 0.21 0.24 0.21 0.22

Kidney IFT NA a NA a 892 897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

IFT-DKN

898 893 895 908

0.114 0.128 0.160 0.166

891 903 905 907

0.296 0.173 0.248 0.223

892 897 900 902

0.503 0.447 0.468 0.495

RPA 205834

898 893 895 908

< 0.05 < 0.05 < 0.05 < 0.05

891 903 905 907

< 0.05 < 0.05 < 0.05 < 0.05

892 897 900 902

< 0.05 0.060 < 0.05 < 0.05

RPA 207048

898 893

< 0.05 < 0.05

891 903

< 0.05 < 0.05

892 897

< 0.05 < 0.05

Isoxaflutole

1297




Animal I.D. mg/kg Animal I.D. mg/kg Animal I.D. mg/kg 895 908

< 0.05 < 0.05

905 907

< 0.05 < 0.05

900 902

< 0.05 < 0.05

Total A 898 893 895 908 average

0.16 0.18 0.21 0.22 0.19

891 903 905 907 average

0.35 0.22 0.30 0.27 0.28

892 897 900 902 average

0.55 0.50 0.52 0.54 0.53

Total B 898 893 895 908 average

0.26 0.28 0.31 0.32 0.29

891 903 905 907 average

0.45 0.32 0.40 0.37 0.38

892 897 900 902 average

0.65 0.61 0.62 0.64 0.63

Liver IFT 898 893 895 908

< 0.05 < 0.05 < 0.05 < 0.05

891 903 905 907

< 0.05 < 0.05 < 0.05 < 0.05

892 897 900 902

< 0.05 < 0.05 < 0.05 < 0.05

IFT-DKN

898 893 895 908

0.499 0.534 0.770 0.696

891 903 905 907

0.879 0.475 1.09 0.941

892 897 900 902

1.72 1.84 1.79 1.70

RPA 205834

898 893 895 908

0.071 0.094 0.082 0.105

891 903 905 907

0.299 0.246 0.231 0.210

892 897 900 902

0.560 0.810 0.750 0.800

RPA 207048

898 893 895 908

< 0.05 < 0.05 < 0.05 < 0.05

891 903 905 907

< 0.05 < 0.05 < 0.05 < 0.05

892 897 900 902

< 0.05 < 0.05 < 0.05 0.136b

Total A 898 893 895 908 average

0.55 0.58 0.82 0.75 0.67

891 903 905 907 average

0.93 0.52 1.1 0.99 0.90

892 897 900 902 average

1.8 1.9 1.8 1.8 1.8

Total B 898 893 895 908 average

0.67 0.73 0.95 0.90 0.81

891 903 905 907 average

1.8 0.82 1.4 1.2 1.2

892 897 900 902 average

2.4 2.7 2.6 2.7 2.6

Total A = sum of IFT and IFT-DKN, expressed as isoxaflutole. Conversion factor of IFT-DKN to isoxaflutole equivalents is 1.00. Residues below LOQ were calculated as being at the LOQ = 0.05 mg/kg. Total B = sum of IFT, IFT-DKN, RPA 205834 and RPA 207048, expressed as isoxaflutole. Conversion factor of IFT-DKN to isoxaflutole equivalents is 1.00; conversion factor for RPA 205834 to isoxaflutole equivalents is 359.3/361.3 = 0.994. Conversion factor of RPA 207048 to isoxaflutole equivalents is 359.3/360.3 = 0.997. Residues below LOQ were calculated as being at the LOQ = 0.05 mg/kg. a NA, assumed to be < 0.05 mg/kg for calculation of total residues b Original value multiplied by a factor 2 to correct for possible degradation of RPA 207048 during frozen storage.

Study 2

A hen feeding study was conducted in the USA in 1995 in order to determine the transfer of isoxaflutole related residues from poultry feed into eggs, muscle, fat and liver [Tew, 1995b, M-192317-01-1]. The study was conducted with 60 laying hens of 1.1–1.8 kg bodyweight (ca. 76 week old) which were distributed in four groups (15 hens per group). Each group was subdivided into three subgroups (A, B, C) containing five hens each. Hens in Group I served as the control group and received a placebo containing cellulose powder. Hens in Groups II, III and IV were orally dosed once a day for 42 days, after the morning egg collection, using gelatine capsules fortified with the appropriate amount of isoxaflutole. Each group's daily dose was calculated and adjusted weekly based

Isoxaflutole

1298

on the previous week's feed consumption for that group (77.9–103.8 g dry matter per hen). Hens in Group II, III and IV were treated with isoxaflutole at actual dose levels of 0.18, 0.54 and 1.8 mg/kg dw feed, respectively, corresponding to 1×, 3×, and 10× dose groups. Equivalent mg/kg bw concentrations were not stated. Eggs were collected each morning and evening throughout the study and pooled by subgroup. Egg collection for analysis began with the afternoon collection prior to initial dose administration (Study Day 0) and continued until the scheduled animal slaughtering for each animal. Each day's PM egg samples were refrigerated overnight until they were pooled with the AM samples. Test animals were slaughtered within 3 hours after receiving the 42nd (final) dose. Samples of liver (entire), muscle (0.2 kg composite of breast and leg) and skin with adhering fat (0.2 kg) were collected. The samples were stored frozen at -15 ºC for a maximum of 83 days for eggs and 64 days for tissues.

Eggs samples were analysed for IFT-DKN only, because preliminary work demonstrated that at high pH (the pH of egg is approx. 9) isoxaflutole degrades rapidly to IFT-DKN. Eggs were analysed using HPLC-UV method #44933. This method is considered valid in the range 0.05–0.25 mg/kg IFT-DKN. Concurrent recoveries for eggs (at 0.05 mg/kg) averaged 92% for IFT-DKN. Residues in control samples were < 0.05 mg/kg.

The tissue samples were analysed for IFT and IFT-DKN using HPLC-MS-MS method RPAC #44996, version 1.0. The recovery of residues was only tested at 0.25 mg/kg level. Concurrent recoveries for tissues (0.05, 0.45, 0.60 or 1.0 mg/kg) ranged between 70–120% for each analyte. It should be noted that separate recovery samples were prepared and analysed: one for IFT and one for IFT-DKN. Isoxaflutole is unstable and rapidly degrades to IFT-DKN. Therefore, IFT recovery was determined by spiking a control sample with IFT only and the recovery was based on the sum of IFT and IFT-DKN from the analysis. Residues in control samples were < 0.05 mg/kg for each analyte.

Egg samples from the Group IV (Days 0, 31, 33, 36, 39 and 41) were analysed for IFT-DKN and no residues (< 0.05 mg/kg) were found. Therefore, no additional egg analyses were performed.

Isoxaflutole related residues in tissues of hens dosed for 42 consecutive days are summarized in Table 60. None of the results presented in these tables have been corrected for recoveries.

Note: Storage stability studies showed that storage periods of 83 days for eggs and 64 days for tissues will not result in breakdown of IFT in tissues or IFT-DKN in eggs or tissues. Results are considered reliable.

Conclusion: The hen feeding study conducted with isoxaflutole shows that residues were mainly transferred to liver. The relationship between IFT-DKN residue levels in liver and the dose level administered is not linear. This may indicate that at the 10× dose level, residue levels have or are beginning to reach saturation.

Table 60 Isoxaflutole related residues in tissues of hens



Group IV 1.8 mg/kg mg/kg in feed (dry matter)

Subgroups mg/kg Subgroups mg/kg Subgroups mg/kg Muscle IFT NA A

B C

< 0.05 < 0.05 < 0.05

A B C

< 0.05 < 0.05 < 0.05

IFT-DKN NA A B C

< 0.05 < 0.05 < 0.05

A B C

< 0.05 < 0.05 < 0.05

Total A A B C average

< 0.1 < 0.1 < 0.1 < 0.1

A B C average

< 0.1 < 0.1 < 0.1 < 0.1

A B C average

< 0.1 < 0.1 < 0.1 < 0.1

Total B A B C average

< 0.1 < 0.1 < 0.1 < 0.1

A B C average

< 0.1 < 0.1 < 0.1 < 0.1

A B C average

< 0.1 < 0.1 < 0.1 < 0.1

Liver IFT NA a A < 0.05 A < 0.05

Isoxaflutole

1299



Group IV 1.8 mg/kg mg/kg in feed (dry matter)

Subgroups mg/kg Subgroups mg/kg Subgroups mg/kg B C

< 0.05 < 0.05

B C

< 0.05 < 0.05

IFT-DKN A B C

0.133 0.159 0.123

A B C

0.327 0.378 0.353

A B C

0.438 0.645 0.588


0.18 0.21 0.17 0.19

A B C average

0.38 0.43 0.40 0.40

A B C average

0.49 0.70 0.64 0.61


0.18 0.21 0.17 0.19

A B C average

0.38 0.43 0.40 0.40

A B C average

0.49 0.70 0.64 0.61

Skin with adhering fat

IFT NA a A B C

< 0.05 < 0.05 < 0.05

A B C

< 0.05 < 0.05 < 0.05

IFT-DKN NA a A B C

< 0.05 < 0.05 < 0.05

A B C

< 0.05 < 0.05 < 0.05


< 0.1 < 0.1 < 0.1 < 0.1

A B C average

< 0.1 < 0.1 < 0.1 < 0.1

A B C average

< 0.1 < 0.1 < 0.1 < 0.1


< 0.2 < 0.2 < 0.2 < 0.2

A B C average

< 0.2 < 0.2 < 0.2 < 0.2

A B C average

< 0.2 < 0.2 < 0.2 < 0.2

Total A = sum of IFT and IFT-DKN, expressed as isoxaflutole. Conversion factor of IFT-DKN to isoxaflutole equivalents is 1.00. Residues below LOQ were calculated as being at the LOQ = 0.05 mg/kg. Total B = sum of IFT, IFT-DKN, RPA 205834 and RPA 207048, expressed as isoxaflutole. Conversion factor of IFT-DKN to isoxaflutole equivalents is 1.00. Residues below LOQ were calculated as being at the LOQ = 0.05 mg/kg. Hen tissues were not analysed for RPA 205038 and RPA 207048, although these metabolites were found in the laying hen metabolism study in hen muscle and hen fat. The sum of IFT and IFT-DKN was therefore multiplied by a factor 1.75 in skin with adhering fat to correct for this omission. Values were than rounded up to 1 figure if below the LOQ. Results in muscle are not corrected because of low expected RPA 207048 levels. Results for liver do not need a correction. a NA, assumed to be < 0.05 mg/kg for calculation of total residues

NATIONAL RESIDUE DEFINITIONS

In the past, the isoxaflutole residue data in plant commodities were generated as the sum of IFT and its metabolites IFT-DKN and IFT-BA using a common-moiety method. Thus, the residue definition for monitoring and dietary risk assessment consisted of the sum of the parent IFT and its metabolites IFT-DKN and IFT-BA, expressed as isoxaflutole. Later on, in the late 1990s the residue data were generated using HPLC-MS-MS methods which allow a separate quantification of isoxaflutole, IFT-DKN and IFT-BA. IFT-BA is a common metabolite for both isoxaflutole and pyrasulfotole (another herbicide). Because, IFT-BA is not considered toxicologically relevant in the EU, Australia, USA and Canada, the residue definition for enforcement has been changed and is now described as the sum of the parent IFT and its metabolite IFT-DKN, expressed as isoxaflutole.

EU

The EU the residue definition for MRL setting and dietary risk assessment for plant and animal commodities is identical and is defined as the sum of isoxaflutole and IFT-DKN, expressed as isoxaflutole.

Isoxaflutole

1300

Australia

Since 2013, the residue definition for both plant and animal commodities is: “The sum of isoxaflutole and 2-cyclopropylcarbonyl-3-(2-methylsulfonyl-4-trifluoromethylphenyl)-3-oxopropanenitrile (i.e. IFT-DKN) expressed as isoxaflutole.

Canada

The residue definition for MRL setting is now the sum of isoxaflutole and IFT-DKN, expressed as isoxaflutole equivalents (EMRL2010-21, 24 September 2010)

USA

The residue definition for MRL setting is now the sum of isoxaflutole and IFT-DKN, expressed as isoxaflutole equivalents (Federal Register / Vol.73, No. 240 of 12 December 2008 and Federal Register / Vol.76, No. 235 of 7 December 2011). Residue definitions for dietary risk assessment are summarized in the Memorandum of 10 July 2008.

Matrix Residues included in Risk Assessment Residues included in Tolerance Expression Plants Maize Isoxaflutole, RPA 202248 Isoxaflutole, RPA 202248 Rotational Crop Rotational crops Isoxaflutole, RPA 202248 Isoxaflutole, RPA 202248 Livestock Ruminant

Poultry Isoxaflutole, RPA 202248, RPA 205834, RPA 207048

Isoxaflutole, RPA 202248

APPRAISAL

Isoxaflutole was scheduled for the evaluation as a new compound by 2013 JMPR at the Forty-fourth Session of the CCPR (2012). Isoxaflutole is a synthetic compound of the isoxazole group of chemicals used as a herbicide. The mode of action of isoxaflutole is the inhibition of the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD), which inhibits pigment formation, causing bleaching of the developing tissues of the target plants. Isoxaflutole controls a wide spectrum of grasses and broadleaf weeds by bleaching emerging or emerged weeds following herbicide uptake via the root system.

The Meeting received information from the manufacturer on identity, metabolism, storage stability, residue analysis, use patterns, residues resulting from supervised trials on sweet corn, chickpeas, glyphosate/HPPD tolerant soya beans, maize, sugar cane and poppy seed, fates of residue during processing, and livestock feeding studies.

Chemical name:

Isoxaflutole

IUPAC: 5-cyclopropyl-4-(2-methylsulfonyl-4-trifluoromethylbenzoyl)-isoxazole

Structural formula:

Metabolites referred to in the appraisal by codes:

IFT-DKN RPA 202248

Isoxaflutole diketonitrile; IUPAC: 3-cyclopropyl-2-[2-mesyl-4-(trifluoromethyl)benzoyl]-3-oxopropanenitrile;

CF3

SO2CH3O

NO

Isoxaflutole

1301

IFT-BA RPA 203328

Isoxaflutole benzoic acid IUPAC: 2-mesyl-4-trifluoromethylbenzoic acid;

IFT-amide (no code)

Isoxaflutole benzamide

IUPAC: 2-mesyl-4-trifluoromethyl benzamide RPA 205834 2-aminomethylene-l-cyclopropyl-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione

RPA 207048 1-cyclopropyl-2-hydroxymethylene-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione

RPA 205568 5-cyclopropyl-1,2-oxazol-4-yl-α,α,α-trifluoro-p-tolyl ketone

No code 4-trifluoromethyl benzoic acid

CH3-C6H4-COOH

Animal metabolism

The Meeting received results of animal metabolism studies in lactating goats and laying hens. Experiments were carried out with [U-14C-phenyl]-isoxaflutole.

Metabolism in laboratory animals was summarized and evaluated by the WHO panel of the JMPR in 2013. Following oral administration in rats, [U-14C-phenyl]-isoxaflutole was rapidly and extensively metabolized yielding nine radioactive fractions in the urine and up to eleven in the faeces. There were no indications of any metabolites resulting from phase II (conjugation) reaction. Parent isoxaflutole was only found in the faeces of the single 100 mg/kg bw high dose group and to a lesser extent also in the urine of this group (together 5.7–8.2% of the administered dose). The major radioactive component (70–85% of the administered dose) in both urine and faeces, as well as in solvent fractions of liver was IFT-DKN for all three dose groups (single 1 mg/kg bw low dose, single 100 mg/kg bw high dose, repeated 1 mg/kg bw low dose).

CF3

OO SO2CH3

CN

CF3

O SO2CH3

OH

CF3

O

NH2

SO2CH3

CF3

OO SO2CH3

NH2

CF3

OO SO2CH3

OH

CF3

O

NO

Isoxaflutole

1302

The parent compound metabolized to IFT-DKN (major pathway) or RPA 205834 and subsequently further oxidized, respectively to IFT-BA (most polar, 0.6–3.5% of the administered dose) or RPA 207048. A third minor metabolic pathway includes the cleavage of the sulfonic acid group to metabolite RPA 205568 (only 1.3–2.0% of the administered dose).

Four lactating goats, orally treated twice daily for 7 consecutive days with [U-14C-phenyl]-isoxaflutole, were sacrificed 23 hours after the last dose. The four goats received low, medium (2) and high actual doses equivalent to 1.1 (goat 1), 10 (goat 2), 13 (goat 3) and 64 (goat 4) ppm dry feed (2.0, 20, 20 and 100 mg ai/kg bw, respectively). Total recovered radioactivity amounted to 97, 88, 78, and 73% of the administered dose in goat 1 to 4, respectively. Radioactivity recovered from urine and faeces ranged from 56% of the administered dose in the high dosed goat (27% in urine, 29% in faeces) to 85% in the low dosed goat (54% in urine, 31% in faeces). Radioactivity in edible tissues and organs ranged from 5.3% of the administered dose in the high dose goat to 11% in the low dose goat. Radioactivity in milk ranged from not detectable in the low dose goat to 0.54–0.60% of the administered dose in the medium to high dosed goats. Radioactivity levels in milk peaked at 0.093–0.095 mg/kg eq in goat 3 at day 4–5, 0.059–0.060 mg/kg eq in goat 2 at day 6–7 and 0.33–0.35 mg/kg eq in goat 4 at day 5–7.

Tissues (goat 3) and milk (goat 2) were subjected to further analysis. The total radioactive residues (TRR) were 2.1 mg/kg eq (liver), 0.90 mg/kg eq (kidney), 0.26 mg/kg eq (muscle), 0.069 mg/kg eq (renal fat), 0.062 mg/kg eq (omental fat) and 0.060 mg/kg eq (milk). Radioactive residues could be extracted with methanol (milk), phosphate buffer pH 7.5 (liver, kidney and fat) or phosphate buffer pH 7.5 in combination with protease treatment (muscle). The extracted radioactive residues in the medium dosed goats amounted to 95% TRR for milk, 98% TRR for liver, 94% TRR for kidney, 76% TRR for muscle, 98% TRR for omental fat and 93%TRR for renal fat.

Parent compound was not found in any of the goat commodities. In goat milk, and all tissues the metabolite IFT-DKN was the most abundant component of the residues in the primary extracts (18–86% TRR or 0.015–1.8 mg/kg eq), followed by the metabolite RPA 207048 (9.1–26% TRR or 0.10–0.016 mg/kg eq, respectively). Muscle contained 18.3% TRR free and 23% conjugated IFT-DKN as well as 9.1% free and 3.4% conjugated RPA 207048; (released as free metabolites by protease treatment). Metabolite RPA 205834 (8.1–18% TRR or 0.005–0.011 mg/kg eq) was only found in milk and fat.

Ten laying hens, orally treated once daily for 14 consecutive days with [U-14C-phenyl]-isoxaflutole, were sacrificed 23 hours after the last dose. Hens were treated at an actual dose rate of 1.1 (group A) or 11 (group B) ppm dry feed. Total recovered radioactivity amounted to 117% and 92% of the administered dose in group A and B, respectively. Radioactivity from the excreta amounted to 112% and 88% of the administered dose for group A and B, respectively. Low levels of radioactivity were recovered in the eggs (0.12–0.15% of the administered dose) or tissues (1.7% in group A and 0.20% in group B).

In the low dose group (A) the concentrations of radioactivity in egg whites were below 0.002 mg/kg eq at all-time points. The levels of radioactivity in egg yolk reached a steady state within 7 days after the first dose (0.022–0.028 mg/kg eq). In the high dose group (B) the concentration of radioactivity in egg whites reached a steady state (0.010–0.015 mg/kg eq) within 4 days. The levels of radioactivity in egg yolk were up to 16 times higher, with a steady state concentration of 0.14–0.15 mg/kg eq within 7 days of exposure.

The highest radioactivity concentrations in edible tissues were found in the liver (0.84 and 0.95 mg/kg eq for dose group A and B, respectively) and kidney (0.055 and 0.16 mg/kg eq, respectively for A and B). Radioactivity in fat and muscle was only observed in the high (B) dose group, being 0.028 and 0.035 mg/kg eq, respectively. Some radioactivity was found in skin (0.008 and 0.068 mg/kg eq in dose group A and B, respectively).

Radioactivity was characterized in tissues and eggs from the high dose group B. Radioactive residues were extracted sequentially by exhaustive extractions using hexane, methanol, acetonitrile, ethyl acetate, acidified methanol and/or water. The primary extractable residues amounted to 97%

Isoxaflutole

1303

TRR for liver, 74% TRR for kidney, 54% TRR for muscle, 93% TRR for fat, 53% TRR for skin, 66% TRR for egg yolk and 50% TRR for egg white. After treatment with protease, another 16%, 29%, 21%, 46% and 20% TRR could be released from kidney, muscle, skin, egg yolk and egg white, respectively. After extensive acid hydrolysis (6 M HCl at 95 ºC for 7 days) another 1.9%, 43%, 7.4% and 20% TRR could be released from kidney, muscle, skin and egg white, respectively.

In egg yolk (0.137 mg/kg eq), the major compounds in the primary extracts represented IFT-DKN (26% TRR) and RPA 205834 (28% TRR). Parent, IFT-BA and RPA 207048 were not found. Exhaustive acid hydrolysis released additional IFT-DKN (17% TRR) from egg yolk. Residues in egg white (0.010 mg/kg eq) and the remaining fractions from egg yolk could not be identified, although two fractions in egg yolk contained considerable radioactivity (10% TRR or 0.014 mg/kg eq in the primary extract and 18% TRR or 0.025 mg/kg eq in the acid hydrolysate).

In hen liver (0.953 mg/kg eq), kidney (0.155 mg/kg eq) and skin (0.068 mg/kg eq), the major compound in the primary extracts represented IFT-DKN (93%, 74% and 37% TRR, respectively). Parent, RPA 207048 and RPA 205834 were not found. IFT-BA was found as minor metabolite in liver and kidney (3.5% TRR and 0.65% TRR, respectively). Protease digestion and exhaustive acid hydrolysis released additional IFT-DKN (18% and 7.4% TRR, respectively) from skin.

In hen muscle (0.035 mg/kg eq) the only compound identified in the primary extracts was RPA 207048 (31%TRR). Parent and RPA 205834 were not found. Exhaustive acid hydrolysis released additional RPA 207048 (17% TRR) as well low levels of IFT-DKN and IFT-BA (5.7% and 5.7% TRR, respectively) from muscle.

In hen fat (0.028 mg/kg eq) the major compounds in the primary extracts were IFT-DKN (29% TRR) and RPA 207048 (21% TRR).

The metabolic pathway of isoxaflutole in livestock involves the opening of the oxazole ring and the formation of the diketo-nitrile derivative (IFT-DKN) or the diketo-amine derivative (RPA 205834). Further degradation occurs through deamination to form the diketo-hydroxy derivative (RPA 207048) or through further cleavage to form the benzoic acid derivative (IFT-BA). Further metabolism involves conjugation of IFT-DKN, RPA 207048 and/or IFT-BA with proteins or acid cleavable compounds.

Parent compound was not found in milk, eggs, goat tissues or hen tissues. This may represent rapid metabolism within the animal or it may represent degradation during frozen storage of the samples or degradation during extraction.

The major compounds identified in goat, hen tissues, milk or eggs are: IFT-DKN, RPA 207048, RPA 205834, conjugated IFT-DKN and conjugated RPA 207048. In goats, metabolites IFT-DKN (18–86% TRR) and RPA 207048 (9.1–26% TRR) were found in all tissues and milk. Metabolite RPA 205834 (8.1–18% TRR) was only found in milk and fat. Conjugated forms of IFT-DKN (23% TRR) and RPA 207048 (3.4% TRR) were only found in goat muscle and free metabolites could be released by protease digestion. In hens, metabolite IFT-DKN (26–93% TRR) was found in eggs and all tissues, except muscle. Metabolite RPA 207048 (21–31% TRR) was only found in muscle and fat. Metabolite RPA 205834 was only found in eggs (28% TRR in egg yolk). Conjugated forms of IFT-DKN (5.7–25% TRR) were found in hen muscle, skin and eggs and free metabolites could be released by protease (skin only) or exhaustive acid hydrolysis. Conjugated forms of RPA 207048 were found in hen muscle only and free metabolites could be released by exhaustive acid hydrolysis. IFT-BA was only found as a minor metabolite in hen liver and kidney (3.5% TRR and 0.65% TRR, respectively) and as conjugate form in hen muscle (5.7% TRR).

The metabolic pathway in ruminants and poultry is identical to the metabolic pathway in rats, although in rats an additional minor pathway to RPA 205568 is found, which is not found in ruminant or poultry. All metabolites identified in livestock are also found in rats.

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Plant metabolism

The Meeting received plant metabolism studies for isoxaflutole on cereals (maize and wheat), pulses/oilseeds (soya beans and poppy seeds) and sugar cane after a pre-plant, crop pre-emergence, crop post-emergence or crop foliar application.

The metabolism of [U-14C-phenyl]-isoxaflutole in outdoor grown maize was studied following pre-plant soil incorporated and pre-emergent applications. Maize forage, grains and fodder were harvested at DAT = 41, 122 and 122/138. Residue levels in maize forage, grains and fodder were 0.20–0.044–0.15 mg/kg eq, respectively, for pre-plant incorporated treatment at 0.21 kg ai/ha and 0.23–0.039–0.12 mg/kg eq, respectively, for pre-emergence treatment after 0.23 kg ai/ha. Residue levels were 0.80–0.15–0.66 mg/kg eq for pre-plant incorporated treatment at 0.66 kg ai/ha and 0.49–0.12–0.53 mg/kg eq for pre-emergence treatment at 1.1 kg ai/ha. Residue levels in maize forage, grains and fodder after pre-plant incorporated treatment were higher than those after pre-emergence treatment at the high dose rate; they were similar at the low dose rates. Exaggerated dose rates exhibited greater phytotoxicity. Radioactivity was characterized in maize from the low dose treatments. The major part of the residues could be extracted with an exhaustive range of solvents: hexane/ethyl acetate, acetonitrile, water pH 5.5, and acidified acetonitrile: 91–99% TRR in forage, 83–87% TRR in grains and 75–79% TRR in fodder. Additional residues could be released by cellulase digestion (3.0–6.5% TRR). Parent isoxaflutole was not found in any of the maize commodities. The major compound identified was free IFT-BA (61–89% TRR) in all maize commodities. Minor metabolites represented conjugated IFT-DKN (0.49–0.53% TRR in forage only) and conjugated IFT-BA (1.1–2.7% TRR in forage and fodder).

In a second study, the metabolism of [U-14C-phenyl]-isoxaflutole was studied in outdoor grown maize following post-emergent application at 0.21 kg ai/ha in the presence of the safener cyprosulfamide. Maize forage and sweet corn were harvested at DAT 75; maize grains and fodder were harvested at DAT = 106. Residue levels in maize forage, sweet corn, maize grains and fodder were 0.13–0.010–0.015–0.10 mg/kg eq, respectively after pre-plant incorporated treatment. These residue levels were similar to those from the pre-plant incorporated and pre-emergence treatments in the previous study. The major part of the residues could be extracted with acetonitrile and water: 93% TRR in forage, 97% TRR in sweet corn, 77% TRR in grains and 88% TRR in fodder. Aqueous fractions underwent base hydrolysis. Parent isoxaflutole was not found in any of the maize commodities. The major compound identified was free IFT-BA (52–63% TRR) in all maize commodities. Other identified metabolites represented free IFT-DKN (4.0–9.8% TRR in sweet corn, fodder and grain) and conjugated IFT-BA (4.1–15% TRR in forage and fodder).

The metabolism of [U-14C-phenyl]-isoxaflutole in outdoor grown wheat was studied following a post-emergent application at 0.055 kg ai/ha onto immature plants (Zadoks 30). Residue levels in wheat hay, straw and grains harvested at DAT 41, 93 and 93/99 were 0.172–0.107–0.058 mg/kg eq, respectively. The major part of the residues (86–96% TRR) could be extracted with acetonitrile/water. Parent isoxaflutole was only found in wheat hay (6.5% TRR). Major metabolites identified were free IFT-BA (65–96% TRR) in all wheat commodities and free IFT-DKN (9.9–21% TRR in forage and straw only). A small fraction of the metabolites in straw might be attributed to conjugates because 3% TRR was released through acid reflux.

The metabolism of [U-14C-phenyl]-isoxaflutole in indoor grown glyphosate/HPPD-tolerant soya was studied following a pre-plant application or a foliar application to plants in full bloom (BBCH 65, 57 days after planting) at 0.331 kg ai/ha. Soya bean forage, hay and seeds were harvested at DAT 74/17, 189/132 and 189/132, respectively, for pre-plant/foliar treatment. Residue levels in soya bean forage, hay and seeds were 0.27–0.49–0.15 mg/kg eq after pre-plant treatment and 13–1.8–0.26 mg/kg eq after foliar treatment. The major part of the residues could be extracted with acetonitrile/water: 91–100% TRR in all soya commodities. The foliar application of isoxaflutole had a significant effect on the metabolic profile. Major compounds identified in the pre-plant application were free IFT-amide (53%, 13% and 8% TRR in forage, hay and seeds, respectively), free IFT-BA (66%, 56% and 27% TRR in seeds, hay and forage, respectively) and free IFT-DKN (13–17% TRR in all soya commodities). Major compounds identified in the foliar application were parent (72% and

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25% TRR in forage and hay, respectively), free IFT-BA (62%, 38% and 6% TRR in seeds, hay and forage, respectively) and free IFT-DKN (18–24% TRR in all soya commodities). Minor metabolites identified in the foliar application were free IFT-amide (3–8% TRR in hay and seeds).

FG 72 soya beans express the hppdPfW336 gene from Pseudomonas fluorescens and the 2mepsps gene derived from maize. These genes confer tolerance to the herbicide isoxaflutole and glyphosate-containing herbicides, respectively, via a modification of the target enzyme which makes the modified enzymes insensitive against the herbicide. Since in the FG72 soya bean variety the tolerance against glyphosate and isoxaflutole is not based on detoxification of the pesticides, the tolerance to glyphosate is not expected to modify the nature and levels of isoxaflutole-derived residues in treated soya beans. Similarly the tolerance to isoxaflutole is not expected to modify the nature and levels of glyphosate residues.

The metabolism of [U-14C-phenyl]-isoxaflutole in outdoor grown poppies was studied following a pre-emergent application (3 days after planting) at 0.11 kg ai/ha in the presence of the safener cyprosulfamide. Residue levels in poppy seeds, seed bolls and straw harvested at DAT 110 were 0.056, 0.78 and 0.72 mg/kg eq, respectively. Most of the radioactivity (92–98% TRR) could be extracted with acetonitrile/water. Parent isoxaflutole was not found in any of the samples. The major compound identified was free IFT-BA (66–94% TRR) in all commodities. Free IFT-DKN was found in low levels (2.1–3.5% TRR) in seed bolls and straw, but not in poppy seeds.

The metabolism of [U-14C-phenyl]-isoxaflutole in outdoor grown sugarcane was studied following a pre-emergent application at 0.210 kg ai/ha or a foliar application (47 days after planting) at 0.133 kg ai/ha. Residue levels in sugarcane plants harvested at DAT 81, 95 and 365 were 0.12–0.15–< 0.01 mg/kg eq, respectively, for pre-emergence treatment and 0.18, < 0.01 and < 0.01 mg/kg eq at DAT 40, 95 and 365, respectively, for foliar treatment. Radioactivity in samples with residues > 0.01 mg/kg eq was characterized. Most of the radioactivity could be extracted with acetonitrile: 66%, 79% and 84% TRR in DAT 40, 81 and 95 samples, respectively. Additional residues (9.5–24% TRR) could be released by exhaustive extraction procedures including reflux with acetonitrile, reflux with 0.1 M HCl and reflux with 0.1 M ammonia. The major compound identified in immature sugarcane (DAT 40, 81 and 95) was IFT-BA (66–93% TRR). Parent isoxaflutole (11% TRR) and IFT-DKN (2.2% TRR) were only found in the foliar treated crop harvested at DAT 40. Since initial acetonitrile extracts were combined with more exhaustive extracts, it is not clear whether the identified compounds are free or conjugated.

The effect of the safener cyprosulfamide on the metabolism of isoxaflutole was investigated in 3 day old maize seedlings grown in nutrient solution. Roots were exposed for 24 h to [U-14C-phenyl]-isoxaflutole alone or in combination with cyprosulfamide. After this period the plants were grown in blank nutrient solution for 3 days. Shoots, seeds and roots were collected and extracted with acetonitrile/water. The effect of the safener cyprosulfamide is a clear reduction of leaf damage (bleaching) and a lower ratio of IFT-DKN to IFT-BA in the shoots.

From these data it is concluded that in cereal grains, seeds of pulses/oilseeds, sugarcanes, forage and fodder of cereals or pulses/oilseeds, metabolite IFT-BA is the only residue identified at significant quantities (52–99% TRR). Parent isoxaflutole (6.5–11% TRR) is only found after post-emergent or foliar treatments in wheat hay or immature sugarcane. Minor metabolites identified were IFT-DKN (2.1–21% TRR in sweet corn, maize grain, immature sugarcane, cereal forage, cereal fodder and poppy straw), conjugated IFT-DKN (0.49–0.53% TRR in maize forage) and conjugated IFT-BA (1.1–15% TRR in maize forage and maize fodder).

Glyphosate/HPPD-tolerant soya has a somewhat different metabolic profile. Major compounds identified in the pre-plant application were free IFT-amide (53%, 13% and 8% TRR in forage, hay and seeds, respectively), free IFT-BA (66%, 56% and 27% TRR in seeds, hay and forage, respectively) and free IFT-DKN (13–17% TRR in all soya commodities). Major compounds identified in the foliar application were parent (72% and 25% TRR in forage and hay, respectively), free IFT-BA (62%, 38% and 6% TRR in seeds, hay and forage, respectively) and free IFT-DKN (18–24% TRR in all soya commodities).

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The first hydrolytic step in the degradation in plants is the opening of the isoxazole ring to form IFT-DKN. Further hydrolytical cleavage of the carbonyl bridge and loss of the complete isoxazole moiety leads to the corresponding benzoic acid derivative (IFT-BA). In glyphosate/HPPD tolerant soya beans loss of the complete isoxazole moiety may also lead to the benzamide derivative (IFT-amide).

Metabolites IFT-DKN and IFT-BA are also found in rat. Metabolite IFT-amide (8–53% TRR) seems to be formed in glyphosate/HPPD tolerant soya beans only. This metabolite was not found in rat.

Environmental fate in soil

The Meeting received information on aerobic degradation in soil, soil photolysis and fate in rotational crops.

Aerobic degradation of [U-14C-phenyl]-isoxaflutole under laboratory conditions was studied at 20 °C in various soil types treated at 0.2 mg ai/kg dry soil (0.20 kg ai/ha). The half-life for isoxaflutole was estimated at 7.6–11 h in clay loam and loamy sand soils, 1.3–2.5 days for sandy loam and clay soils and 4 days for loamy soils. The major metabolites identified were IFT-DKN (max. 52–96% TAR during a period 3–10 days after treatment) and IFT-BA (max. 30–90% TAR during a period of 1–12 months after treatment). IFT-BA levels in clay loam soil were very low (max 7.1% TRR at 7 days). Carbon dioxide was formed from day 1 onwards and these levels increased with time (up to 1.8–37% TRR after 1 year). These study results show that parent isoxaflutole is unlikely to be taken up by crops (and weeds) when applied onto bare soil as pre-plant or pre-emergent application.

Using the data from these soil degradation studies, the half-life for the metabolite IFT-DKN were estimated at 20 days in a sandy loam soil, 25 days in a loam soil, 37 days in a clay soil, 41 days in a clay loam soil and 56 days in a loamy sand soil. The half-life for IFT-BA was estimated at 290 days in clay soil and 980 days in a sandy loam soil. These studies show that metabolites IFT-DKN and IFT-BA are available for take-up by the plants for a considerable period after pre-plant or pre-emergence application depending on the soil type.

Soil photolysis of [U-14C-phenyl]-isoxaflutole was studied in a sandy loam soil, surface treated at 0.645 kg ai/ha and exposed for 31 days to artificial sunlight. The half-life for isoxaflutole (DT50 23 hours) was similar to the one for the dark control (DT50 20 hours). The formation of transformation products specific for photolytic processes was insignificant. The study shows that light has no effect on the degradation of isoxaflutole on soil.

Metabolism of [U-14C-phenyl]-isoxaflutole was investigated in confined rotational crops following pre-plant incorporated soil treatment or pre-emergence treatment. A sandy loam soil was treated at a rate of 0.213 kg ai/ha under outdoor conditions. Rotational crops (radish, lettuce, mustard greens, sorghum and wheat) were sown 34, 123 and 365–375 days after application, representing first, second and third rotation. Total radioactivity for pre-plant incorporated soil treatment ranged from 0.003–0.24 mg/kg eq after first rotation, 0.001–0.030 mg/kg eq after second rotation and 0.001–0.051 mg/kg eq after third rotation. Total radioactivity for pre-emergence application ranged from 0.010–0.126 mg/kg eq after first rotation, < 0.001–0.042 mg/kg eq after second rotation and 0.001–0.030 mg/kg eq after third rotation. Total radioactivity levels above 0.05 mg/kg eq were only found in immature lettuce and sorghum commodities after the first rotation (0.126–0.24 mg/kg eq in sorghum forage, 0.13 mg/kg eq in sorghum fodder, 0.12 mg/kg in sorghum grain, 0.056 mg/kg eq in immature lettuce) and after the third rotation (0.051 mg/kg eq in sorghum forage). Parent isoxaflutole was not found in any of the rotational crops. IFT-BA represented the major compound and was present in the commodities of the first, second and third rotation at levels between < 0.001–0.11 mg/kg eq (6.9–100% TRR). IFT-DKN was only found in radish leaves and sorghum grain at levels up 0.005 mg/kg eq (0.8–27.3% TRR) in the first rotation. RPA 205834 was detected by HPLC-MS-MS in trace amounts (< 0.001 mg/kg eq) in mature lettuce of the first rotation. A fourth metabolite (U1) was found in the commodities of the first, second and third rotation at levels between < 0.001–0.022 mg/kg eq (10–100% TRR). The polarity of this compound and the molecular weight of 192, as

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determined by HPLC-MS-MS, suggests it is a carboxylic acid degradation product of IFT-BA with the structural formula CF3-C6H4-COOH (4-trifluoromethyl benzoic acid).

In a field rotational crop study at two different locations in the USA isoxaflutole was applied as pre-plant or pre-emergent application to maize at 0.154–0.161 kg ai/ha. Rotational crops (soya beans, sugar beets, radishes, turnips, mustard greens, wheat and sorghum) were sown 29/30, 104, 119/120, 151, 166, 180, 365 days after application, representing various rotations of maize. No residues (< 0.01 mg/kg) of parent isoxaflutole, metabolites IFT-DKN or metabolite IFT-BA were found in any of the crops at any of the rotations. Metabolite 4-trifluoromethyl benzoic acid was not analysed, but since its levels are expected to be in the same order of magnitude as IFT-BA, based on the confined rotational crop study, it is not expected to be found at levels > 0.01 mg/kg.

From these data it is concluded that the first hydrolytic step in the aerobic degradation in soil is the opening of the isoxazole ring to form IFT-DKN, which is responsible for the mode of action. Further hydrolytical cleavage of the carbonyl bridge and loss of the complete isoxazole moiety leads to the corresponding benzoic acid derivative (IFT-BA). Light has no effect on the degradation of isoxaflutole on soil. Isoxaflutole has a very short half-life of 8 h to 4 days in soil, and consequently is not found in rotational crops after pre-plant or crop pre-emergence applications. Metabolites IFT-DKN and IFT-BA have long half-lives of 20–56 and 290–980 days, respectively, in soil and consequently are the main metabolites found in plants after pre-plant, crop pre-emergence or crop post-emergence applications. Since metabolites IFT-DKN and IFT-BA were also found after foliar treatments of sugarcane and glyphosate/HPPD tolerant soya beans, it is likely that metabolites IFT-DKN and IFT-BA are also formed in plants. In confined rotational crop studies, a third metabolite (4-trifluoromethyl benzoic acid (CF3-C6H4-COOH)) was found (10–100% TRR), formed by the loss of the methylsulfonyl moiety of IFT-BA. It is not clear whether this metabolite is formed exclusively in the plants or is formed in the soil and taken up by the plants. Metabolite 4-trifluoromethyl benzoic acid was not found in rat.

Methods of Analysis

The Meeting received description and validation data for analytical methods of isoxaflutole related residues in plant and animal commodities.

For plants, a HPLC-MS-MS method was submitted as enforcement/monitoring method for the individual determination of parent and its metabolite IFT-DKN. Plant material was extracted with acidified methanol/water followed by filtration. The Meeting considers validation sufficient for commodities with high acid content, high water content, high starch content and high oil content. The LOQ was 0.01 mg/kg for each analyte.

Several other HPLC-MS-MS methods were submitted for the determination of parent and its metabolites IFT-DKN and IFT-BA in plant material. In some trials a GC-MS method was used, where residues were extracted with methanol and converted into a common moiety IFT-methylbenzoate by hydrolysis and methylation. Most analytical methods were considered fit for purpose with LOQs ranging from 0.01 mg/kg eq for total residues or 0.01–0.03 mg/kg for individual analytes.

For animal commodities, the existing multi-residue method QuEChERS was submitted as enforcement/monitoring method. The Meeting considers this method valid for the individual determination of parent and its metabolite IFT-DKN in all animal commodities. The LOQ was 0.01 mg/kg for milk, eggs, meat, fat, liver and kidney for each analyte.

Three other analytical methods were submitted for the determination of isoxaflutole related residues in milk, eggs or animal tissues. HPLC-UV methods were used for milk and eggs. A HPLC-MS-MS method was used for tissues. Conjugates of IFT-DKN and RPA 207048 were not analysed by these methods. The reported LOQ of 0.05 mg/kg for each analyte in meat, fat, liver and kidney needs to be substantiated by additional data, since only 1–2 recoveries per matrix were provided at this level. Parent is degraded during extraction and is measured as increased IFT-DKN in these methods. Based on the validation data available, the methods are considered suitable for determination of

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parent, IFT-DKN, RPA 205834 and RPA 207048 in milk at 0.02–2.0 mg/kg and 0.05–0.25 mg/kg in eggs and tissues.

Solvents used in the analytical methods were different from the extraction methods used in the metabolism studies. Extraction efficiency, using radiolabelled samples from the metabolism studies, was not verified for any of the analytical methods.

Stability of pesticide residues in stored analytical samples

The Meeting received information on the stability of isoxaflutole, IFT-DKN and IFT-BA in plant commodities or isoxaflutole, IFT-DKN, IFT-BA, RPA 205834 and RPA 207048 in animal commodities in animal commodities stored frozen.

Storage stability studies at -10 ºC and -20 ºC showed that total isoxaflutole residues (sum of parent, IFT-DKN and IFT-BA, measured as common moiety) were stable for at least 11 months in commodities with high protein content (chickpea seeds), 15 months in commodities with high starch content (maize grains) and straw (maize fodder) and at least 20 months in commodities with high water content (sugar canes).

Storage stability studies showed that isoxaflutole converts to IFT-DKN after 3–6 months of storage at -10 ºC in commodities with high acid content (oranges), high water content (sugar canes), high protein content (dry pinto beans) and high oil content (dry soya beans). The metabolite IFT-DKN remained stable for at least 12 months at -10 ºC in commodities with high acid content (oranges), high water content (sugar canes), high protein content (dry pinto beans) and at least 16 months in commodities with high oil content (poppy seeds and dry soya bean seeds). Metabolite IFT-DKN converts to IFT-BA within a period of 23 months at -20 ºC in commodities with high water content (sorghum forage, lettuce and radish leaves), high starch content (radish roots), high protein content (sorghum grain) and straw (sorghum fodder).

Based on storage stability studies at -20 °C in fortified samples of animal commodities the Meeting noted that isoxaflutole was not stable. Isoxaflutole degraded rapidly to IFT-DKN in eggs and degraded after a period of 85 days in milk and muscle. Parent isoxaflutole also degrades to IFT-DKN by the extraction method used for tissue analysis in the feeding studies. Metabolite IFT-DKN is stable for a period of at least 113–130 days in liver, kidney, muscle, fat, milk and eggs. If metabolite IFT-DKN is included in the residue definition, any degradation from isoxaflutole to IFT-DKN is covered by the total residues measured.

Metabolite RPA 205834 is stable for a period of 113–131 days in kidney, muscle, fat and milk. Metabolite RPA 205834 is stable for a maximum storage period of 94 days in liver and degrades significantly thereafter (36% remaining after 130 days). Storage stability of RPA 205834 was not investigated in eggs.

Metabolite RPA 207048 is not stable in any animal commodity. RPA 207048 degrades rapidly in kidney to a level of about 45% which is maintained from 13–115 days. RPA 207048 is stable for a maximum storage period of 28 days in muscle, 40 days in liver and 84 days in fat and thereafter remains stable at a level of about 50% of the original residue level for a period up to 85–113 days. Storage stability of RPA 207048 has not been investigated in milk and eggs. Precautions need to be taken when analysing this metabolite. Besides storage conditions, also extraction conditions are critical.

In case quantitative levels of total residues based on parent, IFT-DKN, RPA 205834 and RPA 207048 are needed, samples of animal origin need to be analysed within 30 days. And even then, metabolite RPA 207048 will be underestimated in kidney.

Definition of the residue

Parent compound isoxaflutole was not found in milk, eggs or livestock tissues. Metabolites found at significant levels in livestock commodities were: IFT-DKN, conjugated IFT-DKN, RPA 205834, RPA 207048, and conjugated RPA 207048. Metabolite IFT-DKN (18–93% TRR) was found in milk, eggs, all goat and hen tissues, except hen muscle. Conjugated forms of IFT-DKN (5.7–25% TRR)

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were only found in goat muscle, hen muscle, hen skin and eggs and the free metabolite could be released by protease digestion (goat muscle and hen skin) and/or exhaustive acid hydrolysis (hen muscle and eggs). Metabolite RPA 205834 (8.1–28% TRR) was found in milk, eggs (yolk) and goat fat in the metabolism studies on goat and hen, but it was also found in milk, bovine liver and bovine kidney in cow feeding study. Metabolite RPA 207048 (9.1–31% TRR) was found in milk, all goat tissues, hen muscle and hen fat. Conjugated forms of RPA 207048 (3.4% TRR) were only found in goat and hen muscle and could be released by protease digestion (goat muscle) or exhaustive acid hydrolysis (hen muscle). Further there seems to be a metabolic shift between RPA 205834 and RPA 207048 between goat and cow. In goat, RPA 207048 seems to be present in all goat tissues and at higher levels than RPA 205834, while in cow RPA 207048 is not found. This metabolic shift might be related to the shorter period between last application and slaughter time for cows (7.5 h) compared to goats (23 h).

Isoxaflutole is easily converted to IFT-DKN. Since no discrimination can be made whether isoxaflutole is metabolized within the animal, or whether it is degraded because of its sensitivity to physical chemical conditions, isoxaflutole needs to be included in the residue definition for enforcement. Any isoxaflutole degraded because of storage or extraction conditions will be measured as IFT-DKN. The sum of IFT and IFT-DKN can therefore serve as the marker residue for enforcement.

The log Kow for isoxaflutole is 2.34. Isoxaflutole and its metabolite IFT-DKN are extracted with acidified aqueous solvents and highest levels of these metabolites are found in the organs kidney and liver. The sum of parent and IFT-DKN is considered not fat soluble.

Apart from isoxaflutole and IFT-DKN other metabolites found at significant levels in livestock commodities were conjugated IFT-DKN, RPA 205834, RPA 207048, and conjugated RPA 207048.

Toxicity of the free IFT-DKN, RPA 205834 and RPA 207048 is considered to be covered by toxicity studies on isoxaflutole since each of the free metabolites was found in the rat high dose group. Free IFT-DKN is the major compound found in rat urine, faeces and solvent fractions of liver, and its toxicity is considered to be similar to that of the parent compound. Since metabolites RPA 205834 and RPA 207048 have similar structures as IFT-DKN and no data are available to conclude that they are of less toxicological significance, each metabolite is considered relevant for the residue definition for dietary risk assessment. Conjugated forms of IFT-DKN and RPA 207048 are considered relevant for dietary exposure, since the free metabolites can be released during the metabolic process in humans. The Meeting proposed to include parent, IFT-DKN, conjugated IFT-DKN, RPA 205834, RPA 207048, and conjugated RPA 207048 in the residue definition for dietary risk assessment of animal commodities.

The analytical method used in the feeding study cannot determine the conjugated forms of IFT-DKN and RPA 207048. For goat muscle, conjugates represent 26.62% TRR and free compounds (IFT-DKN + RPA 205834 + RPA 207048) represent 27.43% TRR. For hen muscle, conjugates represent 22.81% TRR and free compounds represent 31.4% TRR. For hen eggs, conjugates represent 16.8% TRR and free compounds represent 54% TRR. A multiplication factor 2 for goat and hen muscle and 1.3 for hen eggs could be used on the total residues to compensate for this underestimation of dietary exposure.

In primary crops, metabolite IFT-BA is the only residue identified at significant quantities (52–99% TRR). Parent isoxaflutole is only found after post-emergent or foliar treatments in livestock feed commodities: major amounts in glyphosate/HPPD-tolerant soya bean forage and hay (25–72% TRR) and minor amounts (6.5–11% TRR) in wheat hay or immature sugarcane. Minor metabolites identified were IFT-DKN (2.1–21% TRR), conjugated IFT-DKN (0.49–0.53% TRR) and conjugated IFT-BA (1.1–15% TRR). Metabolite IFT-amide (8–53% TRR) seems only to be formed in glyphosate/HPPD tolerant soya bean (seeds, forage, hay).

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Metabolite IFT-BA is the major residue and is relevant for consideration in the residue definition for enforcement. However, IFT-BA can also arise in plant commodities as a result of treatment with pyrasulfotole. For this reason IFT-BA cannot be used as a marker for isoxaflutole.

The only other compounds relevant for the residue definition for enforcement are the parent and IFT-DKN. The sum of IFT and IFT-DKN can therefore serve as the marker residue for enforcement.

Apart from isoxaflutole and IFT-DKN, other metabolites found at significant levels in plant commodities are IFT-BA (feed commodities and tolerant soya beans) and IFT-amide (tolerant soya beans only). Assessment of additional toxicological data on IFT-BA demonstrated that metabolite IFT-BA is considerably less toxic then the parent compound. Thus, from a toxicological point of view it is not necessary to include IFT-BA in the residue definition. Metabolite IFT-amide is not found in rat. But since IFT-amide is present at lower levels than IFT-BA in soya bean seeds and because of its structural similarity with IFT-BA, IFT-amide is considered not relevant for the residue definition.

The Meeting recommended the following residue definition for isoxaflutole:

Definition of the residue for compliance with the MRL and for dietary risk assessment for plant commodities: sum of isoxaflutole and isoxaflutole diketonitrile, expressed as isoxaflutole.

Definition of the residue for compliance with the MRL for animal commodities: sum of isoxaflutole and isoxaflutole diketonitrile, expressed as isoxaflutole.

The Meeting considers the residue not fat soluble.

Definition of the residue for dietary risk assessment for animal commodities: sum of isoxaflutole, isoxaflutole diketonitrile, RPA 205834 (2-aminomethylene-l-cyclopropyl-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione) and RPA 207048 (1-cyclopropyl-2-hydroxymethylene-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione), including their conjugates, expressed as isoxaflutole.

Results of supervised residue trials on crops

The total residue values selected for maximum residue level recommendations and dietary intake are based on the sum of isoxaflutole and IFT-DKN. In case a common moiety method is used for analysis also the IFT-BA metabolite is included. Since the relative molecular weight of IFT-DKN is identical to that of isoxaflutole, no molecular weight conversion is needed. Soil type and the addition of the safener cyprosulfamide affect the residue levels in plant commodities. Residue trials were conducted in a range of soil types, which included those with the longest half-lives.

Since IFT-DKN is included in the residue definition, any degradation from isoxaflutole to IFT-DKN is covered by the total residues measured. Since IFT-BA is not part of the residue definition, any degradation from IFT-DKN to IFT-BA means an underestimation of the original residue present in the sample. Therefore, the Meeting takes only those trials into account, where samples have been stored for a maximum of 16 months (commodities with high oil content) or 12 months (all other commodities). Also trials where IFT-BA is below LOQ can be taken into account, because it indicates that no degradation of IFT or IFT-DKN to IFT-BA occurred.

Sweet corn (corn-on-the-cob)

Field trials involving sweet corn were performed in Spain, Italy, Greece, Portugal, Germany, France, Netherlands and the United Kingdom.

Critical GAP for sweet corn in France is for a single post sowing pre-emergence treatment (BBCH 00–08) at 0.099 kg ai/ha, where the safener cyprosulfamide is added. Trials from Spain, Italy, Germany, France, Netherlands and the UK (0.099–0.10 kg ai/ha, growth stage BBCH 0–06, cyprosulfamide added) matched this GAP. For sweet corn harvested at BBCH 79, total residues were: < 0.02 (10) mg/kg (n=10).

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Several trials, where isoxaflutole was applied at the same dose rate at a later growth stage (BBCH 13–14), as well as a metabolism study in sweet corn following a post-emergence treatment at 0.21 kg ai/ha, confirmed the non-residue situation.

The Meeting estimated a maximum residue level of 0.02* mg/kg on sweet corn (corn-on-the-cob). The Meeting estimated an STMR of 0 mg/kg.

Pulses

Field trials involving chick-peas (dry) were performed in Australia.

Critical GAP for chick-peas in Australia is a single post plant crop, pre-emergence application at 0.075 kg ai/ha. In trials from Australia (1× 0.075 kg ai/ha post planting pre-emergent) matching this GAP, total residues were: < 0.01 (3) and < 0.01a mg/kg (n=4) with a common moiety method (including IFT-BA). The superscript (a) indicates the addition of an adjuvant. The addition of which did not result in a difference in total residue levels.

An additional four trials at the same locations using a single post-plant crop emergence application at a higher application rate of 0.15 kg ai/ha, as well as a metabolism study in poppy seeds after post-plant crop pre-emergence treatment at 0.11 kg ai/ha, confirmed the no residue situation.

The Meeting estimated a maximum residue level of 0.01* mg/kg on chickpea, dry and an STMR of 0 mg/kg.

Field trials involving glyphosate/HPPD tolerant soya beans (FG72) (dry) were performed in the USA and Canada.

No authorised uses were available for glyphosate/HPPD tolerant soya beans (FG72). The Meeting agreed that no recommendations could be set for soya beans.

Maize

Field trials involving maize were performed in Spain, Italy, Greece, Portugal, Germany, France, Netherlands, the United Kingdom, the USA and Canada.

Critical GAP for maize in France is a single early post emergence treatment up to 3 leaf stage of the crop (BBCH 13) at 0.099 kg ai/ha with the addition of the safener cyprosulfamide. In field trials from Spain, Italy, Greece, Portugal, Germany, France, the Netherlands and the United Kingdom (1× 0.10 kg ai/ha, BBCH 13, with cyprosulfamide) matching this GAP, total residues for maize grain harvested at BBCH 89 were: < 0.02 (14) mg/kg.

The GAP for maize in the USA is a single pre-emergence treatment at 0.16 kg ai/ha without the addition of the safener cyprosulfamide or a single early post-emergence treatment at 0.105 kg ai/ha up to 2 leaf-collar growth stage of the crop (GS 12) with the addition of the safener cyprosulfamide. In field trials from the USA (1× 0.15–0.17 kg ai/ha, pre-emergent, no adjuvants) matching the first GAP, total residues for maize grains harvested at maturity were < 0.02 (17) mg/kg. In field trials from the USA (1× 0.13 kg ai/ha, post-emergent GS 12, with safener cyprosulfamide, no adjuvants) matching the second GAP, total residues for maize grains harvested at maturity were < 0.03 (17) mg/kg.

A metabolism study with mature maize grains at 0.21 kg ai/ha after pre plant or post-emergence application did not confirm the non-residue situation.

The Meeting estimated a maximum residue level of 0.02* mg/kg and an STMR of 0.02 mg/kg.

Sugar cane

Supervised residue trials on sugar cane were conducted in Australia, Mexico and Brazil.

There were no authorised uses available for Mexico and Brazil.

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Critical GAP for sugar cane in Australia is a single soil directed application at 0.15 kg ai/ha when the sugar cane was at least 0.75 m high and with a PHI of 19 weeks without adjuvant. In field trials from Australia (1× 0.150 kg ai/ha, PHI 133–166 days) matching this GAP, total residues were < 0.01 (2) mg/kg with a common moiety method (including IFT-BA).

Two additional trials, at the same locations, at 1× 0.225 kg ai/ha and a PHI of 133–166 days, two trials in Brazil using two applications at 0.150 kg ai/ha and a PHI of 92–95 days and a metabolism study in mature sugar cane after soil directed application to emerged plants at 0.133 kg ai/ha, confirmed the no residue situation.

The Meeting estimated a maximum residue level of 0.01* mg/kg on sugar cane and an STMR of 0 mg/kg.

Poppy seed

Field trials involving poppy seed were performed in France, Spain, Germany, Netherlands and Hungary.

Critical GAP for poppy seed in Spain is for a single pre-emergence application at 0.060 kg ai/ha without the safener cyprosulfamide. There were no trials matching this GAP.

Critical GAP for poppy seed in Hungary is for a single pre-emergence application at 0.11 kg ai/ha with the safener cyprosulfamide. In field trials from Germany, the Netherlands and Northern France (1× 0.10 kg ai/ha, pre-emergence, with cyprosulfamide) matching this GAP, total residues poppy seeds were < 0.02 (3) mg/kg.

A metabolism study in poppy following post-plant crop pre-emergence treatment at 0.11 kg ai/ha confirmed the no residue situation.

The Meeting estimated a maximum residue level of 0.02* mg/kg in poppy seeds and an STMR of 0 mg/kg.

Legume animal feeds

Field trials involving chick-pea forage were performed in Australia.

Critical GAP for chickpeas in Australia is a single post plant crop pre-emergence application at 0.075 kg ai/ha with a PHI of 6 weeks for use as forage. There were no trials matching this GAP.

Field trials involving chick-pea fodder were performed in Australia.

Critical GAP for chick-peas in Australia is a single post plant crop pre-emergence application at 0.075 kg ai/ha. In trials from Australia (1× 0.075 kg ai/ha, post planting pre-emergent) matching this GAP, total residues were: < 0.01 (3) and < 0.01a mg/kg (n=4) with a common moiety method (including IFT-BA). The superscript (a) indicates the addition of adjuvant. The addition of which did not result in a difference in total residue levels.

An additional trial at the same location using a single post-plant pre-emergence application at a higher application rate of 0.20 kg ai/ha did not confirm the non-residue situation.

The Meeting estimated a maximum residue level of 0.01* mg/kg on chickpea fodder, a median residue of 0.01 mg/kg and a highest residue of 0.01 mg/kg for livestock dietary burden calculations.

Forage and fodder of cereal grains and grasses

Field trials involving maize forage were performed in Spain, Italy, Greece, Portugal, Germany, France, the Netherlands, the United Kingdom, the USA and Canada.

Critical GAP for maize in France is a single early post-emergence treatment up to 3 leaf stage of the crop (BBCH 13) at 0.099 kg ai/ha with the addition of the safener cyprosulfamide. No PHI is mentioned. High total residues ranging from 1.3–16 mg/kg eq were found immediately after treatment (DAT=0). However, the Meeting considers DAT=0 irrelevant for maize forage harvest and considers

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growth stage BBCH 16 or 33 (3 nodes with 6 leaves) the earliest grazing time for maize forage. In field trials from Spain, Italy, Greece, Portugal, Germany, France, Netherlands and the United Kingdom (1× 0.099–0.10 kg ai/ha, BBCH 13, with cyprosulfamide) matching the French GAP, total residues for maize forage harvested at BBCH 19 or 33–35 (DAT 40–41) were < 0.02 (11) and 0.34 mg/kg (n=12).

Critical GAP for maize in the USA is a single early post-emergence treatment at 0.105 kg ai/ha up to 2 leaf-collar growth stage of the crop (BBCH 14 or 32) with the addition of the safener cyprosulfamide, with or without the addition of an adjuvant. The pre-harvest interval is 45 days. In field trials from the USA (1× 0.13 kg ai/ha, PHI 43–45 days, with safener cyprosulfamide, no adjuvants) matching this GAP, total residues for early maize forage were < 0.03 (15) mg/kg.

The Meeting agreed that the dataset matching French GAP could be used to estimate a median residue of 0.02 mg/kg and a highest residue of 0.34 mg/kg for livestock dietary burden calculations.

Field trials involving maize fodder were performed in Spain, Italy, Greece, Portugal, Germany, France, Netherlands, the United Kingdom, the USA and Canada.

Critical GAP for maize in France is a single early post emergence treatment up to 3 leaf stage of the crop (BBCH 13) at 0.099 kg ai/ha with the addition of the safener cyprosulfamide. In field trials from Spain, Italy, Greece, Portugal, Germany, France, Netherlands and the United Kingdom (1× 0.099–0.10 kg ai/ha, BBCH 13, with cyprosulfamide) matching this GAP total residues for maize fodder harvested at BBCH 79 (sweet corn fodder) were < 0.02 (13) mg/kg.

Critical GAP for maize in the USA is a single pre-emergence treatment at 0.16 kg ai/ha without the addition of the safener cyprosulfamide or a single early post-emergence treatment at 0.105 kg ai/ha up to 2 leaf-collar growth stage of the crop (i.e. GS 12) with the addition of the safener cyprosulfamide, each with or without the addition of an adjuvant. In field trials from the USA (1× 0.15–0.17 kg ai/ha, pre-emergent, no adjuvants) matching the first GAP, total residues for maize fodder harvested at dent stage to maturity were < 0.02 (17) mg/kg. In field trials from the USA (1× 0.13 kg ai/ha, post-emergent GS 12, with safener cyprosulfamide, no adjuvants) matching the second GAP, total residues for maize fodder harvested at maturity were < 0.03 (14) mg/kg.

A metabolism study with mature maize fodder at 0.21 kg ai/ha after post-emergence application did not confirm the non-residue situation.

The Meeting estimated a maximum residue level of 0.02* mg/kg, a median residue of 0.02 mg/kg and a highest residue of 0.02 mg/kg for livestock dietary burden calculations.

Miscellaneous forage and fodder crops

Supervised residue trials on sugar cane tops/fodder were conducted in Australia.

The Meeting estimated a maximum residue level of 0.01* mg/kg on sugar cane fodder, a median residue of 0 mg/kg and a highest residue of 0.01 mg/kg for livestock dietary burden calculations.

Residues from rotational crops

Total residues above 0.01 mg/kg eq are not expected in rotational crops.

Fate of residues during processing

Processing studies with isoxaflutole were undertaken for soya beans. However, since no MRLs could be set for soya beans, no processing factors are needed.

Residues in animal commodities

The Meeting estimated the dietary burden of isoxaflutole residues on the basis of the livestock diets listed in the FAO manual appendix IX (OECD feedstuff table). Calculation from highest residue and

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STMR (some bulk commodities) provides the levels in feed suitable for estimating maximum residue levels, while calculation from STMR values from feed is suitable for estimating STMR values for animal commodities. Commodities used in the dietary burden calculation are maize grains, maize forage, chickpea fodder, maize forage, maize fodder and sugar cane tops.

Dietary burden calculations for beef cattle, dairy cattle, broilers and laying poultry are provided in Annex 6. A mean and maximum dietary burden for livestock, based on isoxaflutole use, is shown in the table below. Animal dietary burden for isoxaflutole total residues, expressed as ppm of dry matter diet

US EU AU JP overall

max max max max max beef cattle 0.146 0.685 0.688 0.017 0.688 dairy cattle 0.394 0.518 0.688 0.436 0.688 a, b poultry broiler 0.017 0.016 – 0.016 0.017 poultry layer 0.017 0.102 – 0.018 0.102 c, d mean mean mean mean mean

beef cattle 0.026 0.045 0.048 0.017 0.048 dairy cattle 0.034 0.038 0.048 0.036 0.048 a, b poultry broiler 0.017 0.016 – 0.016 0.017 poultry layer 0.017 0.022 – 0.018 0.022 c, d

a Highest mean and maximum dietary burden suitable for maximum residue level and STMR estimates for mammalian meat b Highest mean and maximum dietary burden suitable for maximum residue level and STMR estimates for milk c Highest mean and maximum dietary burden suitable for maximum residue level and STMR estimates for poultry meat d Highest mean and maximum dietary burden suitable for maximum residue level and STMR estimates for eggs

Livestock feeding studies

The Meeting received a feeding study on lactating cows and laying hens. Total residues in animal commodities for enforcement are defined as the sum of isoxaflutole and IFT-DKN, expressed as isoxaflutole equivalents. Total residues for dietary risk assessment are defined as the sum of isoxaflutole, IFT-DKN, RPA 205834 and RPA 207048, and their conjugates, expressed as isoxaflutole equivalents.

Four groups of four lactating Holstein cows were dosed once daily via capsules at levels of 0.0, 4.7, 14.4 and 45.5 ppm parent compound in dry weight feed for 42 consecutive days. Milk was collected throughout the study and tissues were collected on day 42 within 7.5 h after the last dose. Milk was not analysed for RPA 207048, but the level of RPA 207048 has been estimated based on relative levels to the other metabolites in the metabolism study. Residues found at the 4.7 ppm dose level are summarized in the residues section below.

Four groups of 15 laying hens were dosed once daily via capsules at levels of 0.0, 0.18, 0.54 and 1.8 ppm parent compound in dry weight feed for 42 consecutive days. Eggs were collected throughout the study and tissues were collected on day 42 within 3 h after the last dose. Eggs and hen tissues were not analysed for RPA 205834 and RPA 207048, but the level of these metabolites has been estimated based on relative levels to the other metabolites in the metabolism study. Residues found at the 0.18 ppm dose level are summarized in the residue section below.

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Residues in animal commodities

Cattle

For maximum residue level estimation, the highest residue in the tissues and milk were calculated by interpolating the maximum dietary burden (0.688 ppm) between the relevant feeding levels (0–4.7 ppm) from the dairy cow feeding study and using the highest tissue concentrations based on the residue definition for enforcement from individual animals within those feeding groups and using the mean milk concentration from those feeding groups (see table below).

The STMR values for the tissues and milk were calculated by interpolating the mean dietary burden (0.048 ppm) between the relevant feeding levels (0–4.7 ppm) from the dairy cow feeding study and using the mean tissue and milk concentrations based on the residue definition for dietary risk assessment from those feeding groups (see table below).

Total residues (mg/kg eq) in Feed level

(ppm) for milk residues

Total residues (mg/kg eq) in milk

Feed level (ppm) for tissue residues

Muscle Liver Kidney Fat

Maximum residue level—beef or dairy cattle (residue definition for enforcement) Feeding study a 4.7 NA 4.7 NA 0.82 0.22 NA Dietary burden and residue estimate

0.688 0 0.688 0 < 0.1 < 0.1 0

STMR—beef or dairy cattle (residue definition for dietary risk assessment) Feeding study b 4.7 NA 4.7 NA 0.81 0.29 NA Dietary burden and residue estimate

0.048 0 0.048 0 < 0.2 < 0.2 0

NA not analysed, because highest dose level of 45.5 ppm showed residues below or just above the LOQ a highest residues for tissues and mean residues for milk b mean residues for tissues and mean residues for milk

The Meeting estimated a maximum residue level for total isoxaflutole residues of 0.01* mg/kg in meat (from mammals other than marine mammals), mammalian fats (except milk fats), milks, and 0.1 mg/kg in mammalian edible offal. The residue in animal commodities is considered not fat soluble.

The Meeting estimated an STMR for total isoxaflutole residues of 0 mg/kg in meat (from mammals other than marine mammals), mammalian fats (except milk fats) and milks, and 0.2 mg/kg in mammalian edible offal.

Poultry

For maximum residue level estimation, the high residue in the tissues and eggs were calculated by interpolating the maximum dietary burden (0.102 ppm) between the relevant feeding levels (0–0.18 ppm) from the laying hen feeding study and using the highest tissue and egg concentrations based on the residue definition for enforcement from individual animals within those feeding groups (see table below).

The STMR values for the tissues and eggs were calculated by interpolating the mean dietary burden (0.022 ppm) between the relevant feeding levels (0–0.18 ppm) from the laying hen feeding study and using the mean tissue and egg concentrations based on the residue definition for dietary risk assessment from those feeding groups (see table below).

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Total residues (mg/kg eq) in Feed level

(ppm) for egg residues

Total residues (mg/kg eq) in egg

Feed level (ppm) for tissue residues

Muscle Liver Kidney Fat

Maximum residue level—poultry broilers or layers (residue definition for enforcement) Feeding study a 0.18 NA 0.18 NA 0.21 – NA Dietary burden and residue estimate

0.102 0 0.102 0 0.12 – 0

STMR—poultry broilers or layers (residue definition for dietary risk assessment) Feeding study b 0.18 NA 0.18 NA 0.19 – NA Dietary burden and residue estimate

0.022 0 0.022 0 < 0.1 – 0

NA not analysed, because highest (1.8 ppm) and medium (0.54 ppm) dose level did not show residues a highest residues for tissues and eggs b mean residues for tissues and eggs

The Meeting estimated a maximum residue level for isoxaflutole total residues of 0.2 mg/kg in poultry edible offal, 0.01* mg/kg in poultry meat, poultry fats and poultry eggs. The residue in animal commodities is considered not fat soluble.

The Meeting estimated an STMR for isoxaflutole total residues of 0.1 mg/kg in poultry edible offal, 0 mg/kg in poultry meat and poultry fats, and eggs.

RECOMMENDATIONS

On the basis of the data from supervised trials the Meeting concluded that the residue levels listed below are suitable for establishing maximum residue limits.

Definition of the residue for compliance with the MRL and for dietary risk assessment for plant commodities: sum of isoxaflutole and isoxaflutole diketonitrile, expressed as isoxaflutole.

Definition of the residue for compliance with the MRL for animal commodities: sum of isoxaflutole and isoxaflutole diketonitrile, expressed as isoxaflutole.

The Meeting considers the residue not fat soluble.

Definition of the residue for dietary risk assessment for animal commodities: sum of isoxaflutole, isoxaflutole diketonitrile, RPA 205834 (2-aminomethylene-l-cyclopropyl-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione) and RPA 207048 (1-cyclopropyl-2-hydroxymethylene-3-(2-mesyl-4-trifluoromethylphenyl)-propane-1,3-dione), including their conjugates, expressed as isoxaflutole.

Summary of recommendations

CCN Commodity name MRL mg/kg

STMR mg/kg

HR mg/kg

VO 0447 Sweet corn (corn-on-the-cob) 0.02* 0 –

VD 0524 Chick-pea (dry) 0.01* 0 –

GC 0645 Maize 0.02* 0.02 –

GS 0659 Sugar cane 0.01* 0 –

SO 0698 Poppy seed 0.02* 0 –

AL 0524 Chick-pea fodder 0.01* 0.01 (median residue)

0.01 (highest residue)

AS 0645 Maize fodder 0.02* 0.02 0.02

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CCN Commodity name MRL mg/kg

STMR mg/kg

HR mg/kg

(median residue) (highest residue)

MM 0095 Meat (from mammals other than marine mammals)

0.01* 0 –

MO 0105 Edible offal (Mammalian) 0.1 0.2 –

MF 0100 Mammalian fats (except milk fats) 0.01* 0 –

ML 0106 Milks 0.01* 0 –

PM 0110 Poultry meat 0.01* 0 –

PF 0111 Poultry fats 0.01* 0 –

PO 0111 Poultry, Edible offal of 0.2 0.1 –

PE 0112 Eggs 0.01* 0 –

Feedstuffs

Crop Feedstuff Highest Residue

STMR or STMR-P

Corn, field (maize)

forage/silage (forage)

0.34 0.02

Corn, field stover 0.02 0.02

Corn, sweet (maize)

Forage 0.34 0.02

Corn, sweet stover 0.02 0.02

Pea (chickpea)

Hay (fodder)

0.01 0.01

Sugar cane Tops/forage 0.01 0

DIETARY RISK ASSESSMENT

Long-term intake

The International Estimated Daily Intakes (IEDI) of for isoxaflutole was calculated from recommendations for STMRs for raw and processed commodities in combination with consumption data for corresponding food commodities. The results are shown in Annex 3 of the 2013 JMPR Report.

The IEDI of in the 13 GEMS/Food cluster diets, based on the estimated STMRs were in the range 0–1% of the maximum ADI of 0.02 mg/kg bw. The Meeting concluded that the long-term intake of residues of isoxaflutole from uses considered by the Meeting is unlikely to present a public health concern.

Short-term intake

Since no ARfD is considered necessary, no short-term intake assessment is considered necessary. The Meeting concluded that the short-term intake of residues of isoxaflutole from uses considered by the Meeting is unlikely to present a public health concern.

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Beedle, EC & Dallstream, KA

2010 Title: Balance Pro 480 SC and Glyfos—Magnitude of the residue in/on soya beans. Source & Testing Facility: Bayer CropScience, Stilwell, KS, USA.Date: 13 May 2010 Report No: RAISP010; MRID: 48108112.Published: No Claim for Data protection: Yes. GLP: yes

M-364166-01-1

Billian, P & Krusell, L

2010a Title: Determination of the residues of AE 0001789 and isoxaflutole in/on poppy after spraying of Isoxaflutole & Cyprosulfamide SC 480 in the field in Spain. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 25 February 2010 Report No: 09-2085. Published: No. Claim for Data protection: Yes. GLP: yes

M-360114-02-1

Billian, P & Krusell, L

2010b Title: Determination of the residues of AE 0001789 and isoxaflutole in/on poppy after spraying of Isoxaflutole & Cyprosulfamide SC 480 in the field in France (North), Germany and the Netherlands—amendment no 1. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 19 October 2010 Report No: 08-2094. Published: No Claim for Data protection: Yes. GLP: yes. Amendment to report 08-2094 dated 4 December 2012

M-360144-02-1

Billian, P & Reineke, A

2010 Title: Determination of the residues of AE 0001789 and isoxaflutole in/on poppy after spraying of Isoxaflutole & Cyprosulfamide SC 480 in the field in France (South) and Spain—amendment no 0001. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 8 March 2010 Report No: 08-2095. Published: No Claim for Data protection: Yes. GLP: yes. Amendment to report 08-2095 dated 9 December 2009.

M-240821-01-1

Burr, CM 1996 Title: Herbicides: Isoxaflutole Route of Degradation (Aerobic Metabolism) in one Soil. Source & Testing Facility: Rhone-Poulenc Agriculture Limited, Ongar, Essex, United Kingdom. Date: 12 August 1996 Report No: P94/094, code B003826, DocMap 201142; MRID: 45658804 Published: No.. Claim for Data protection: Yes. GLP: yes.

M-286476-01-1

Class, T 2007 Title: Independent laboratory validation of Bayer CropScience method no. 01021 for the determination of residues of isoxaflutole and its metabolite RPA 202248 in/on plant material by LC/MS/MS. .Source: BCS. Testing Facility: PTRL Europe, Ulm, Germany. Date: 30 March 2007 Report No: P/B 1182G; study no P612077503. Published: No. Claim for Data protection: Yes. GLP: yes.

M-162558-01-1

Corgier, MM, Robin, JM & Plewa, AP

1994 Title: 14C-RPA201772 Hydrolysis. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France. Date: 2 May 1994 Report No: R&D/CRLD/AN/9415453, study no 93-180, code R002384. Published: No. Claim for Data protection: Yes. GLP: yes.

M-162794-01-1

Corgier, MM & Plewa, AP

1995 Title: 14C-RPA201772 (isoxaflutole) Photodegradation in water. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France. Date: 13 January 1995 Report No: R&D/CRLD/AN/9416822; study no 94-11, code R002507. Published: No. Claim for Data protection: yes. GLP: yes.

M-162115-01-1

Cousin, JA 1993a Title: RPA201772 active ingredient—Physical and chemical characteristics—Part A: Physical characteristics. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France Lyon, France. Date: 10 September 1993 Report No: R&D/CRLD/AN/9316753, Study no 93-129; code R002177. Published: No. Claim for Data protection: Yes. GLP: yes.

M-162129-01-2

Cousin, JA 1993b Title: RPA201772 active ingredient—Physical and chemical characteristics—Part B: pH and Dissociation Constant. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France; Date: 4 October 1993 Report No: R&D/CRLD/AN/9316835; study no 93-129; Published: No. Claim for Data protection: Yes GLP: yes.

M-162137-01-1

Cousin, JA 1993c Title: RPA201772 active ingredient—Physical and chemical characteristics—Part C: Solubilities. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France. Date: 11 October 1993 Report No: R&D/CRLD/AN79316923 Study no 93-129; code R002177; MRID 43573205. Published: No. Claim for Data protection: Yes. GLP: yes.

M-162435-01-1

Cousin, JA 1994a Title: RPA201772 active ingredient—Physical and chemical characteristics—Part F: Vapour pressure. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France; Date: 28 March 1994 Report

Isoxaflutole

1319

Code Author Year Title, Institute & Report reference No: R&D/CRLD/AN/9415227. Study number 93-129, code R002324; MRID: 43573208.Published: No. Claim for Data protection: Yes. GLP: yes.

M-213139-02-1

Cousin, JA 1994b Title: RPA 201772 active ingredient Physical and chemical characteristics Part A: Physical characteristics amendment. Source & Testing Facility: Rhone-Poulenc, Secteur Agro, Lyon, France. Date: 25 May 1994 Report no R&D/CRLD/AN/9415719; Study no 93-129; code C022461. Published: No. Claim for Data protection: Yes. GLP: yes.

M-162438-03-1

Cousin, JA 1995a Title: RPA201772 active ingredient—Physical and chemical characteristics—Part D: Octanol / water partition coefficient—Amendment no 1. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Lyon, France. Date: 18 January 1995 Report No: R&D/CRLD/AN/9515091; study no 93-129, doc nr 437493, code R014775. Published: No. Claim for Data protection: Yes. GLP: yes. Amendment no 1 to original report. R&D/CRLD/AN 9415236, 2 March 1994.

M-162149-02-1

Cousin, JA 1995b Title: Amendment No. 1—RPA201772 active ingredient—Physical and chemical characteristics—Part E: Stability. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France. Date: 5 October 1995 Report No: R&D/CRLD/AN/9516351 Study no 93-129, Code C022462. Published: No. Claim for Data protection: Yes. GLP: yes. Amendment no 1 to Original report R&D/CRLD/AN/9316985, 15 November 1993.

M-368661-01-1

Dallstream, KA & Fischer, DR

2010 Title: Balance Pro 480 SC—Magnitude of the residue in/on soya beans. Source & Testing Facility: Bayer CropScience, Stilwell, KS, USA. Date: 7 May 2010 Report No: RAISP006; MRID 48108110. Published: No. Claim for Data protection: Yes. GLP: yes.

M-284383-01-1

Davis, L & Keats, A

1999a Title: Isoxaflutole and metabolites (RPA202248 and RPA203328)—Formulation: Balance 750 g/kg WG (EXP31130A)—Mareeba, QLD—Australia 1998—Residues in sugarcane and associated portions: Expressed juice, bagasse and forage. Source: Rhone-Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 2 April 1999 Report No: AK99009; Protocol no 95h118eR. Published: No. Claim for Data protection: Yes. GLP: no.

M-284347-01-1

Davis, L & Keats, A

1999b Title: Isoxaflutole and metabolites (RPA202248 and RPA203328)—Formulation: Balance 750 g/kg WG (EXP31130A)—Atherton, QLD—Australia 1998—Residues in sugarcane and association portions: Expressed juice, bagasse and forage. Source: Rhone Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 4 April 1999 Report No: AK99010 Protocol no 95h118dR. Published: No. Claim for Data protection: Yes. GLP: no.

M-284356-01-1

Davis, L & Keats, A

1999c Title: Isoxaflutole and metabolites (RPA202248-RPA203328)—Formulation: Balance 750 g/kg WG—Murwillumbah, QLD—Australia 1998—Residues in sugarcane and associated portions: Expressed juice, bagasse & forage. Source: Rhone-Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 6 April 1999 Report No: AK99011 Protocol no AUS95h118cR. Published: No. Claim for Data protection: Yes. GLP: no.

M-284369-01-1

Davis, L & Keats, A

1999d Title: Isoxaflutole and metabolites (RPA202248-RPA203328)—Formulation: Balance 750 g/kg WG—Rocky Point, QLD—Australia 1998—Residues in sugarcane and associated portions: Expressed juice, bagasse & forage. Source: Rhone-Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 8 April 1999 Report No: AK99012, Protocol no AUS95h118fR. Published: No. Claim for Data protection: Yes. GLP: no.

M-284408-01-1

Davis, L & Keats, A

1999e Title: Isoxaflutole and metabolites (RPA202248-RPA203328)—Formulation: Balance 750 g/kg WG—Jardine, QLD—Australia 1997—Residues in sugarcane and associated portions: Expressed juice, bagasse & forage. Source: Rhone-Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 8 April 1999 Report No: AK99017; Protocol no AUS95h118bR. Published: No. Claim for Data protection: Yes. GLP: no.

M-284324-01-1

Davis, L & Keats, A

1999f Title: Isoxaflutole and metabolites (RPA202248-RPA203328)—Formulation: Balance 750 g/kg WG—Bundaberg, QLD—Australia 1997—Residues in sugarcane juice, bagasse & forage. Source: Rhone-Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 10 April 1999 Report No AK99013; Protocol no AUS95h118aR. Published: No. Claim for Data protection: Yes. GLP: no.

M-425905-02-1

Ellis, A 2012 Title: Determination of residues of isoxaflutole in the forage, seed and stubble of chickpeas following a fallow application of Balance 750 WG at rates of 75 and 150 g ai/ha and also following the fallow application along with an application at sowing—amendment. Source & Testing Facility: Bayer CropScience Residue Laboratory, Eight Mile Plains, QLD, Australia. Date: 26 April 2012 Report No: BCS-0370. Published: No. Claim for Data protection: Yes. GLP: yes. Amendment to study BCS-0370, dated 6 February 2012.

Isoxaflutole

1320

Code Author Year Title, Institute & Report reference M-158351-01-1

Ferreira, EM 1994 Title: RPA201772—Soil photolysis. Source & Testing Facility: Rhone-Poulenc Agriculture Limited, Ongar, Essex, United Kingdom. Date: 4 February 1994 Report No: P93/122, Code R000311. Published: No. Claim for Data protection: Yes. GLP: yes.

M-158435-01-1

Ferreira, EM, Jones, MK & Newby, SE

1994 Title: RPA201772: Aerobic soil metabolism. Source & Testing Facility: Rhone-Poulenc Agriculture Ltd., Ongar, Essex, United Kingdom. Date: 13 October 1994 Report No: P92/332, code R000347. Published: No. Claim for Data protection: Yes. GLP: yes.

M-240822-01-1

Ferreira, EM, Bullus, CM, Jones, MK;& Simmonds, MB

1996 Title: RPA 201772: Rate of Degradation under Aerobic Conditions in Three Soil Types. Source & Testing Facility: Rhone-Poulenc Agriculture Limited, Ongar, Essex, United Kingdom. Date: 10 January 1996 Report No: P94/093, code B003827, RPA Document 201036; MRID: 45658805. Published: No. Claim for Data protection: Yes. GLP: yes.

M-368662-01-1

Fischer, DR 2010 Title: Balance Pro 480 SC—Magnitude of the residue in soya bean processed commodities and aspirated grain fractions. Source & Testing Facility: Bayer CropScience, Stilwell, KS, USA. Date: 7 May 2010 Report No: RAISP005; MRID 48108113. Published: No. Claim for Data protection: Yes. GLP: yes.

M-285889-01-1

Fischer, DR & Helfrich, KK

2007 Title: Isoxaflutole plus AE 0001789 480 SC—Magnitude of the isoxaflutole residue in/on field corn. Source: Bayer CropScience, Research Triangle Park, NC, USA. Testing Facilities: Bayer CropScience, Stilwell, KS, USA; Battelle AgriFood, Columbus, OH, USA. Date: 16 March 2007 Report No: RAUBP008; MRID: 47114035. Published: No. Claim for Data protection: Yes. GLP: yes.

M-238729-01-1

Gough, STD 2000 Title: Isoxaflutole: Magnitude of residues in sugarcane after application of EXP 31130A to crops grown in Mexico. Source & Testing Facility: Aventis CropScience, Research Triangle Park, NC, USA. Date: 9 November 2000 Report No: 99717930; Agredoc Number B003075. Published: No. Claim for Data protection: Yes. GLP: yes.

M-189749-01-1

Guesnet, JL, Jendrzejczak, NH, Maestracci, MP, Ott, M & Vidal, JG

1994 Title: RPA201772—NMR, IR, MS and UV-visible spectra. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France. Date: 10 March 1994 Report No: R&D/CRLD/AN/9415238 studies no 93–184, code R014773. Published: No. Claim for Data protection: Yes.

M-213160-01-1

Guillet, M, Diot, R & Le Gren, I

1995 Title: Isoxaflutole and/or metabolites—Analytical method for the determination of residues in animal products–Method AR 109-95(E). Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France. Date: 12 April 1995 Report No: R&D/CRLD/AN/bd/9515587, code C022471. Published: No. Claim for Data protection: Yes. GLP: yes.

M-162883-01-1

Hampton, RE & Pettaway, J

1995a Title: 14C-RPA201772: Metabolic fate and distribution in corn (Zea mays L.)—(171–4 Nature of residue—Plants). Source: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA Testing Facility: A&L Great Lakes Laboratories, Fort Wayne, IN, USA. Date: 13 February 1995 Report No: EC-93-293 code R002551 Published: No. Claim for Data protection: Yes GLP: yes.

M-192308-01-1

Hampton, RE & Pettaway, J

1995b Title: 14C-RPA201772: Accumulation study on confined rotational crops. Source: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Testing Facilities: American Agricultural Services, Inc, Lucama, NC, USA. AGVISE, Inc., Northwood, ND, USA. Date: 28 December 1995 Report No: EC 93-242, code R016766 & 439564; MRID 43904839. Published: No. Claim for Data protection: Yes. GLP: yes.

M-192325-01-1

Hampton, RE 1996 Title: Supplemental report: 14C-RPA201772: Accumulation study on confined rotational crops. Source & Testing Facility: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Date: 27 November 1996 Report No: EC 93-242, code R016770; MRID: 43904839. Published: No. Claim for Data protection: Yes. GLP: yes.

M-262714-01-1

Hudson, JR, Vincent, RJ, Wells-Knecht, KJ, Seymour, RJ, Vialaneix, JP & Jones, M.

1996 Title: Method of analysis for the determination of isoxaflutole (RPA201772) and its metabolites (RPA202248, RPA207048, and RPA205834) in animal tissues. Source & Testing Facility: Rhone-Poulenc Ag Company, USA. Date: 29 January 1996 Report No: none. Published: No. Claim for Data protection: Yes. GLP: no.

M-213302-01-1

Kaune, A 2002 Independent Laboratory Validation of the method of analysis for the determination of isoxaflutole (RPA 201772) and its metabolites (RPA 202248 and RPA 205834) in milk, eggs, liver, kidney, muscle and fat tissues—version 2.0. Source & Testing Facility: Bayer CropScience GmbH, Frankfurt am Main, Germany. Date: 13 December 2002 Report No. 02F028, Code C027428. Published: No. Claim for Data protection: Yes. GLP: yes.

Isoxaflutole

1321

Code Author Year Title, Institute & Report reference M-442915-01-1

Klasmeier, BJ 2012 Title: Stability of residues of isoxaflutole and its metabolite RPA 202248 during frozen storage in several raw agricultural commodities. Source: Bayer Crop Science, Research Triangle Park, NC, USA. Testing Facility: Morse Laboratories, Sacramento, CA, USA. Date: 7 December 2012 Report No: RAISP012. Published: No. Claim for Data protection: Yes. GLP: yes.

M-360799-01-1

Klempner, A 2009 Title: Metabolism of [phenyl-UL-14C] isoxaflutole in poppies. Source & Testing Facility: Bayer CropScience AG, Monheim am Rhein, Germany. Date: 17 December 2009 Report No: MEF-09/499; Study no M1731775-1. Published: No. Claim for Data protection: Yes. GLP: yes.

M-366215-01-1

Klempner, A 2010 Title: Storage stability of isoxaflutole residues in poppy seeds and straw. Source & Testing Facility: Bayer CropScience AG, Monheim am Rhein, Germany. Date: 18 March 2010 Report No: MEF-10/038, Study no M9991930-0. Published: No. Claim for Data protection: Yes. GLP: yes.

M-170844-01-1

Lappin, GJ 1995a Title: (14C)-RPA201772: Absorption, distribution, metabolism and excretion following repeated oral administration to the laying hen. Source: Rhone-Poulenc Secteur Agro, Sophia Antipolis Cedex, Valbonne, France. Testing Facility: Corning Hazleton (Europe), Harrogate, North Yorkshire, United Kingdom. Date: 30 November 1995 Report No: 198/77-1011, study no 198/77, code R005378. Published: No. Claim for Data protection: Yes. GLP: yes.

M-166744-01-2

Lappin, GJ 1995b Title: (14C)-RPA201772: Absorption, distribution, metabolism and excretion following repeated oral administration to the dairy goat. Source: Rhone-Poulenc Secteur Agro, Sophia Antipolis, Valbonne, France. Testing Facility: Corning Hazleton (Europe), Harrogate, North Yorkshire, United Kingdom. Date: 27 December 1995 Report No: 198/78-1011, Study no 198/78. Published: No. Claim for Data protection: Yes. GLP: yes.

M-213162-01-1

Le Gren, I 1995a Title: Isoxaflutole and/or metabolites—Analytical method for the determination of residues in animal products—Complementary report no 1—method AR109-95(E). Source & Testing Facility: Rhone-Poulenc Secteur Agro, Centre de Recherche de la Dargoire, Lyon, France. Date: 19 May 1995 Report No: R&D/CRLD/AN/fb/9515783, code C022472. Published: No. Claim for Data protection: Yes. GLP: yes. Complementary to report R&D/CRLD/AN/bd/9515587 Guillet et al, 1995, M-213160-01-1.

M-262409-01-1

Le Gren, I 1995b Title: Isoxaflutole: Determination of residues in egg. Source & Testing Facility: Rhone-Poulenc Secteur Agro, Lyon, France. Date: 18 July 1995 Report No: i&D/CRLD/AN/bd/9516020, Doc no 438730. Published: No. Claim for Data protection: Yes. GLP: no.

M-213164-01-1

Le Gren, I 1995c Title: Isoxaflutole and/or metabolites—Analytical method for the determination of residues in animal products—Complementary report no. 2—method AR109-95(E). Source & Testing Facility: Rhone-Poulenc Secteur Agro, Lyon, France. Date: 20 September 1995 Report No: R&D/CRLD/AN/fb/9516279 code C022473. Published: No. Claim for Data protection: Yes. GLP: yes. Complementary to report R&D/CRLD/AN/bd/9515587. Guillet et al, 1995, M-213160-01-1.

M-166734-01-2

Lopes, A, Russell, DR, Smitley, CA & Oden, KN

1998 Title: Method of analysis for the determination of isoxaflutole (RPA 201772) and its metabolites (RPA 202248 and RPA 205834) in milk, eggs, liver, kidney, muscle and fat tissues—version 2.0. Source & Testing Facility: Rhone Poulenc AG Company, Research Triangle Park, NC, USA. Date: 4 March 1998 Report No: RPAC 45532. Published: No. Claim for Data protection: Yes. GLP: No.

M-192281-01-2

Lowder, JF 1996 Title: Isoxaflutole: Storage stability of residues in dairy cow and poultry matrices. Source & Testing Facility: Rhone-Poulenc Ag Company, Research Triangle Park, USA. Date: 19 November 1996 Report No: EC-96-338, File No. 45178. Published: No. Claim for Data protection: Yes. GLP: yes.

M-432364-01-1

Lynch, A; Keats A

1999a Title: Isoxaflutole; Formulation—Balance; Pittsworth, QLD—Australia 1999; Residues in chickpea. Source: Rhone-Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 20 December 1999 Report No: AK99054, protocol no 99NST26. Published: No. Claim for Data protection: Yes .GLP: no.

M-432374-01-1

Lynch, A; Keats A

1999b Title: Isoxaflutole; Formulation—Balance; Biniguy, NSW—Australia 1999; Residues in chickpea. Source: Rhone-Poulenc Rural, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 22 December 1999 Report No: AK99056, protocol no 99NST26. Published: No. Claim for Data protection: Yes. GLP: no.

M-432434-01-1

Lynch, A & Keats A

2000a Title: Isoxaflutole; Formulation—Balance; Horsham, VIC—Australia 1999; Residues in chickpea. Source: Aventis CropScience, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 15 February 2000 Report No: AQ00001, Protocol no 99h011. Published: No. Claim for Data protection: Yes.

Isoxaflutole

1322

Code Author Year Title, Institute & Report reference GLP: no.

M-432441-01-1

Lynch, A & Keats A

2000b Title: Isoxaflutole; Formulation—Balance; Horsham, VIC—Australia 1999; Residues in chickpea. Source: Aventis CropScience, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 16 February 2000 Report No: AQ00002, protocol no 99h015. Published: No. Claim for Data protection: Yes. GLP: no.

M-432444-01-1

Lynch, A & Keats A

2000c Title: Isoxaflutole; Formulation —Balance; Bruce Rock—Australia 1999; Residues in chickpea. Source: Aventis CropScience, Australia. Testing Facility: A-Quant Laboratories, Brisbane, Australia. Date: 16 February 2000 Report No: AQ00003, protocol no 99h010. Published: No. Claim for Data protection: Yes. GLP: no.

M-268739-01-2

Meyer, BN & Ripperger, R

2006 Title: The metabolism of [phenyl-UL-14C]-Isoxaflutole in corn with post-emergence application. Source & Testing Facility: Bayer CropScience, Stilwell, KS, USA. Date: 27 February 2006 Report No: MEUBY003. Published: No. Claim for Data protection: Yes. GLP: yes.

M-240663-01-1

Mickelson, K 1999a Title: Magnitude of Residue of EXP 31130A in Field Corn. Source: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Testing Facilities: American Agricultural Services, Inc., Cary, NC, USA. Centre Analytical Laboratories, Inc, State College, PA, USA. Date: 5 November 1999 Report No: AA98714495; RPAC file 45979, code B003643; MRID: 45655904. Published: No. Claim for Data protection: Yes. GLP: yes.

M-240664-01-1

Mickelson, K 1999b Title: Magnitude of Residue of EXP 31130A in/on Processed Fractions of Field Corn. Source: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Testing Facilities: American Agricultural Services, Inc., Cary, NC, USA. Centre Analytical Laboratories, Inc, State College, PA, USA. Texas A&M University, Bryan, TX, USA. Date: 2 December 1999 Report No: AA98715447; code B003644; RPAC file no 45990, MRID: 45655905. Published: No. Claim for Data protection: Yes. GLP: yes.

M-240710-01-1

Mickelson, K 2000 Title: Isoxaflutole: Field Rotational Crop Study Using Balance WDG Herbicide. Source: Aventis CropScience, Research Triangle Park, NC, USA. Testing Facilities: American Agricultural Services, Inc., Cary, NC, USA. Centre Analytical Laboratories Inc, State College, PA, USA. Date: 7 January 2000 Report No: AA97712512, code B003705, File no 45997. Published: No. Claim for Data protection: Yes. GLP: yes.

M-020184-01-1

Nandihalli, UB 1995 Title: EXP 30953B/field corn/magnitude of residue (US93702R): Analytical method for the determination of residue of RPA 201772, RPA 202248, and RPA 203328 in corn forage, silage, grain, and fodder. Bayer CropScience Method No. 00473.Source & Testing Facility: Hazleton Wisconsin Inc., Madison, WI, USA. Date: 3 January 1995 Report No: HWI 6224-215, Study no US93702R. Published: No. Claim for Data protection: Yes. GLP: no.

M-089371-01-1

Nandihalli, UB 1996 Title: Freezer storage stability of RPA 201772 in field corn samples. Source: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Testing Facility: Corning Hazleton, Inc., Madison, WI, USA. Date: 7 November 1996 Report No: CHW 6224-223, code R016771. Published: No. Claim for Data protection: Yes. GLP: yes.

M-368555-01-1

Nguyen, T 2010 Title: The metabolism of [phenyl-UL-14C] isoxaflutole in soya bean with pre-plant and post-emergent application. Source & Testing Facility: Bayer CropScience, Stilwell, KS, USA. Date: 10 May 2010 Report No: MEISP002, MRID: 48108105. Published: No. Claim for Data protection: Yes. GLP: yes.

M-366025-01-1

Noss, G; Reineke, A

2010 Title: Determination of the residues of AE 0001789 and isoxaflutole in/on poppy after spraying of isoxaflutole & cyprosulfamide SC 480 in the field in Hungary.. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 25 March 2010 Report No: 09-2022; Lynx-ID: RAUBL007. Published: No. Claim for Data protection: Yes. GLP: yes.

M-282394-02-1

Schoening, R 2007 Title: Analytical method 01021 for the determination of residues of isoxaflutole and its metabolite RPA 202248 in/on plant material by HPLC-MS/MS. Amendment no 1. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany Date: 30 January 2007 Report No: MR-06/167, project ID P682 06 4720. Published: No. Claim for Data protection: Yes. GLP: yes. Amendment no 1 to original report: MR-06/167 dated 9 January 2007.

M-280607-01-1

Schoening, R & Wolters, A

2006 Title: Modification M001 of analytical method 00985 for the determination of residues of isoxaflutole and its metabolites AEB197278-AE0540092 (RPA202248) and AE0317309-AEB197555 (RPA203328) in/on corn plant material by HPLC-MS/MS. Bayer CropScience Method No. 00985/M001. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 13 November 2006 Report No: MR-06/038. Project ID: P672064703. Published: No.

Isoxaflutole

1323

Code Author Year Title, Institute & Report reference Claim for Data protection: Yes. GLP: yes.

M-210791-01-1

Schulte, W 2002 Title: Effects of safener AE 0001789 on metabolism of isoxaflutole (IFT) (RPA 201772) in maize. Source & Testing Facility: Aventis CropScience GmbH, Frankfurt am Main, Germany. Date: 16 April 2002 Report No: WS-2002-1; code C021218. Published: No. Claim for Data protection: Yes. GLP: no.

M-192304-01-2

Shaffer, SR 1995 Title: Independent laboratory validation of the Rhone-Poulenc methods entitled, "Method of analysis for the determination of isoxaflutole and its metabolites RPA203328, RPA202248, and RPA205834 in milk" and "Method of determination for isoxaflutole (RPA201772) and its metabolite RPA202248 in/on bovine and poultry tissues”, version 2.0. Source: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Testing Facility: Horizon Laboratories, Inc., Columbia, MO, USA. Date: 22 December 1995 Report No: RPAC File #44920.EC-95-323/HL REPORT #10144. Published: No. .Claim for Data protection: Yes. GLP: yes.

M-368825-01-1.

Stoughton, SM 2010a Title: Analytical method for the determination of residues of isoxaflutole (RPA 201772) and its metabolite RPA 202248 in plant matrices using LC/MS/MS. Residue Analytical Method No. IS-004-P10-01. Source & Testing Facility: Bayer CropScience, Stilwell, KS, USA. Date: 18 February 2010 Report No: IS-004-P10-01. Published: No. Claim for Data protection: Yes. GLP: yes.

M-368826-01-1

Stoughton, SM 2010b Title: Analytical method for the determination of residues of isoxaflutole (RPA 201772) and its metabolite RPA 202248 in plant matrices using LC/MS/MS. Residue Analytical Method No. IS-004-P10-02. Source: Testing Facility: Date: 12 May 2010 Report No: IS-004-P10-02. Published: No. Claim for Data protection: Yes. GLP: yes.

M-368828-01-1

Stoughton, SM 2010c Title: Validation of Bayer method IS-004-P10-01 analytical method for the determination of residues of isoxaflutole (RPA 201772) and its metabolite RPA 202248 in plant matrices using LC/MS/MS. Source & Testing Facility: Bayer CropScience, Stilwell, KS, USA. Date: 12 May 2010 Report No: RAISP007; MRID: 48108107. Published: No. Claim for Data protection: Yes. GLP: yes.

M-192311-01-1

Tew, EL 1995a Title: Isoxaflutole: magnitude of residues in milk and tissues of lactating dairy cows. Source & Testing Facility: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Date: 27 December 1995 Report No: US95704R; File No.: 44961 code R016767. Published: No. Claim for Data protection: Yes. GLP: yes.

M-192317-01-1

Tew, EL 1995b Title: Isoxaflutole: magnitude of residues in tissues and eggs of laying hens. Source & Testing Facility: Rhone-Poulenc Ag Company, Research Triangle Park, NC, USA. Date: 28 December 1995 Report No: RPAC US95705R. File No.: 44981, code R016768. Published: No. Claim for Data protection: Yes. GLP: yes.

M-287038-01-1 and M-411759-02-1 (English translation)

Tornisielo, VL 2000a Title: Determinação de resíduo de Provence 750 WG (Isoxaflutole) em colmos de cana de açúcar (Assis-SP). Source: Aventis CropScience Brasil Ltda., Sao Paulo, Brazil. Testing Facility: Universidade de Sao Paulo, Centro de Energia Nuclear na Agricultura, Laboratorio de Ecotoxicologica, Piracicaba, Brazil. Date: 10 July 2000 Report No: USP BRA98R27-2; Project no AV01/00. Published: No. Claim for Data protection: Yes. GLP: no. English translation Title: Isoxaflutole; 75 WG; sugar cane; Brazil; BBA.

M-287041-01-1 and M-411752-02-1 (English translation)

Tornisielo, VL 2000b Title: Determinação de resíduo de Provence 750 WG (Isoxaflutole) em colmos de cana de açúcar (Cosmópolis-SP). Source: Aventis CropScience Brasil Ltda., Sao Paulo, Brazil. Testing Facility: Universidade de Sao Paulo, Centro de Energia Nuclear na Agricultura, Laboratorio de Ecotoxicologica, Piracicaba, Brazil. Date: 10 July 2000 Report No: USP BRA98R27-1; Project no AV02/00. Published: No. Claim for Data protection: Yes. GLP: no. English translation: Title: Isoxaflutole; 75 WG; sugar cane; Brazil; BBA.

M-211498-01-1

Unsworth, RH 1999 Title: (14C)-RPA 201772: Metabolism in sugarcane. Source & Testing Facility: Rhone-Poulenc Agriculture Ltd., Ongar, Essex, United Kingdom. Date: 15 September 1999 Report No: 10316, code C026486, RPA document 201746. Published: No. Claim for Data protection: Yes. GLP: yes.

M-211481-01-1

Unsworth, RH & Clarke, DE

2000 Title: (14C)-Isoxaflutole: Metabolism in wheat. Source & Testing Facility: Aventis CropScience, Ongar, Essex, United Kingdom. Date: 21 March 2000 Report No: 16862; code C026477, document 202392. Published: No. Claim for Data protection: Yes. GLP: yes.

M-286216-02-1

Vaughn, FC & Cosgrove D

2007 Title: Magnitude of residues in/on corn treated with one application of the herbicide IFT/1789 480 SC with a 110 day phi for grain. Source: Bayer Crops Science, Regina, UK. Testing Facility: ALS Canada Inc, Edmonton, Canada. Date: 2 April 2007 Report No: 07BAY36.REP, code RAUBP011. Published: No. Claim for Data protection: Yes. GLP: yes.

M-286215- Wickremesinhe, 1998a Title: Method of analysis for the determination of isoxaflutole (RPA 201772) and

Isoxaflutole

1324

Code Author Year Title, Institute & Report reference 01-1 E its metabolites (RPA 202248 and 203328) in raw agricultural commodities and

processed foods (Revision 1). Source: Rhone-Poulenc Agro, Lyon, France. Testing Facility: Centre Analytical Laboratories, Inc., State College, PA, USA. Date: 29 December 1998 Report No: CAL #019-03 (revision 1). Published: No. Claim for Data protection: Yes. GLP: no.

M-240820-01-1

Wickremesinhe, E

1998b Validation of method of analysis for the determination of isoxaflutole (RPA 201772) and its metabolites in raw agricultural commodities and processed foods by LC/MS/MS. Source: Rhone-Poulenc Ag Company (RPAC). Testing Facility: Centre Analytical Laboratories Inc (CAL), State College, PA, USA. Date: 22 October 1998 Report No RPAC EC-98-431, CAL 019-008, code B003824. Published: No. Claim for Data protection: Yes. GLP: no.

M-454397-01-1

Winter O & Amann S

2013 Validation of the BCS-method-01300/M009 (based on QuEChERS) for the determination of residues of isoxaflutole and its metabolite RPA 202248 in animal tissues. Source: Bayer CropScience, Monheim am Rhein, Germany. Testing facility: Eurofins Agrosciences Services Chem, Hamburg, Germany, Date 13 May 2013 Report no S12-00056 (BAY-12078V), code RAISX027. Published: No. Claim for Data protection: Yes. GLP: yes.

M-285005-01-1

Wolters, A 2007a Title: Determination of the residues of AE 0001789, isoxaflutole and BYH 18636 in/on corn after spraying of AE 0001789 & Isoxaflutole (480 SC) and AE 0001789 & BYH 18636 (450 SC) in the field in southern France, Spain and Italy. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 8 March 2007 Report No: RA-2616/06. Published: No. Claim for Data protection: Yes. GLP: yes.

M-285014-01-1

Wolters, A 2007b Title: Determination of the residues of AE 0001789, isoxaflutole, and BYH 18636 in/on corn after spraying of AE 0001789 & Isoxaflutole (480 SC) and AE 0001789 & BYH 18636 (450 SC) in the field in northern France, United Kingdom and Germany. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 8 March 2007 Report No: RA-2615/06. Published: No. Claim for Data protection: Yes. GLP: yes.

M-284416-01-1

Wolters, A & Erler, S

2007a Title: Determination of the residues of AE 0001789, BYH 18636 and isoxaflutole in/on corn after spraying of BYH 18636 & IFT & AE 0001789 (465 SC) in the field in southern France, Italy and Spain. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 26 February 2007 Report No: RA-2511/06. Published: No. Claim for Data protection: Yes. GLP: yes.

M-284423-01-1

Wolters, A & Erler, S

2007b Title: Determination of the residues of AE 0001789, BYH 18636 and isoxaflutole in/on corn after spraying of BYH 18636 & IFT & AE 0001789 (465 SC) in the field in northern France, Germany and United Kingdom. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 26 February 2007 Report No: RA-2510/06. Published: No. Claim for Data protection: Yes. GLP: yes.

M-281611-01-1

Zimmer, D 2006 Title: Determination of the residues of AE 0001789 and isoxaflutole in/on corn after spraying of AE 0001789 & isoxaflutole (480 SC) in the field in France, Spain, Italy, Greece and Portugal. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 21 December 2006 Report No: RA-2588/05. Published: No. Claim for Data protection: Yes. GLP: yes.

M-282674-01-1

Zimmer, D & Wieland, K

2007 Title: Determination of the residues of AE 0001789 and isoxaflutole in/on corn after spraying of AE 0001789 & isoxaflutole (480 SC) in the field in Germany, Northern France, United Kingdom and the Netherlands. Source & Testing Facility: Bayer CropScience AG, Monheim, Germany. Date: 23 January 2007 Report No: RA-2587/05. Published: No. Claim for Data protection: Yes. GLP: yes.

ISOXAFLUTOLE (268) · Isoxaflutole was scheduled for evaluation as a new compound by 2013 JMPR at...

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