Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data

Page **1** of **18 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY** 

**WASHINGTON, D.C. 20460**

**OFFICE OF CHEMICAL SAFETY AND** 

**POLLUTION PREVENTION MEMORANDUM**  **DATE:** February 29 , 201 2 **SUBJECT: Tolclofos**  **- methyl**  **: Non**  **- food Use**  **Fo r**  **Seed Treatment on Various** 

**Crops PC Code:**  128905 **DP Barcode s**  **:** D 395999 and D397276 **Decision No.:** 443921 **Registration No. :**  NA **Petition No.:** NA **Regulatory Action:** NA **Risk Assessment Type:** Single chemical **Case No.:** NA T**XR No.:** NA **CAS No.:** 57018
- 04
- 9 **MRID No.:** 48341956 , 48627101 and 48341957 **40 CFR:** NA

**REVIEWER:** Sheila Piper, Chemist

Risk Assessment Branch VI Health Effects Division (HED, 7509P) **THROUGH:** Felecia Fort, Chief

Risk Assessment Branch VI Health Effects Division (HED, 7509P) **TO:** Lisa Jones /Mary Waller, Risk Management Team 21 Fungicide Branch Registration Division Risk Management Division

**Executive Summary** Tolclofos

- methyl is a seed treatment agent to control a number of soil

- borne fungi including Rhizoctonia solani in cotton and other crops. In this study , the radiolabeled test substance, [phenyl

14 C]tolclofos

- methyl, was applied to soy bean, corn, lettuce, cucumber, radish, and cotton seeds. The study was conducted to determine if the seed treatment use of tolclofos

- methyl could be designated for non

- food
.Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 2 of 18 The registrant, Valent U.S.A. Corporation met with EPA on October 2, 2008 to discuss tolclofos

- methyl radiotracer studies which were conducted to support the non

- food classification and the required toxicology data requirements which were based on Valent’s review of existing data and their interpretation of the 40 CFR Part 158 requirements for nonfood uses. Subsequently , HED’s Chemistry Science Advisory Council (ChemSAC) met on December 3, 2008 to discuss Valent’s proposal for non

- food use classification of the seed treatment uses of tolclofos

- methyl on various crops and crop groups . ChemSAC concluded if all TRR results are less than 5 ppb (based on memorandum of Chemistry SAC
- HED
- 7509C titled “Classification of Seed Treatments as Food or Nonfood Uses ” dated Oct 28, 1999) , then the proposed seed treatment use s of tolclofos

- methyl on various crops can be classified as non

- food. To determine the total radioactive residue (TRR), aliquots of samples were combusted in a biological oxidizer followed b y liquid scintillation counting (LSC) of the aliquot vials. HPL C was used to determine the radiochemical purity of the test substance be fore and after seed treatment. The limit of quantification (LOQ) is 0.0 03 ppm. The method was adequate for data collection based on acceptable concurrent method recoveries
. Freezer storage stability

data for soy

bean, corn, cotton, cucumber, lettuce and radish are adequate. The dates that the samples were planted, collected and anal yz ed were less than 30 day s; therefore, a freezer storage stability study is not needed. The freezer storage period before the extraction anal ys is of treated radish tops was 13.7 months. The submitted TRR data on various crops are adequate to satisfy data requirements and supports the seed treatment uses being classified as non

- food uses. The results showed that the soy

bean, corn, lettuce , cucumber , radish and cotton plants grown for seed treated with [phenyl

14 C]tolclofos

- methyl at 10 or 50 g.ai/100kg did not take up radiolabeled test substance residue ( < 5 ppb). The radish tops showed residue levels above 5 ppb at the rate of 50 g/100 kg. Only the radish top sample s grown from seeds showed TRR residues above 0.005 ppm. The radish tops grown from seeds contained residues at 0.02

– 0.03 ppm. Since the radish tops grown for seeds showed residues above 0.005 ppm, additional characterization of the residues was provide d. Two treated and one control crop samples were extracted and anal yzed. The treated samples were radish tops (TRR = 0.029 ppm and TRR = 0.020 ppm) from replicate plots TR1 and TR2, respectivel y. The control radish tops grown from untreated seeds were spiked with radiolabeled tolclofos

- methyl and anal yz ed to determine the efficiency of the extraction method. The radish tops grown for seeds were extracted and anal yz ed b y HP LC . Both acetonitrile and aqueous extracts showed some polar radioactive residues, but did not contain any detectable amount of tolclofos

- methyl. There are currently

no established or proposed tolerances for tolclofos

- methyl in the United States as the proposed seed treatment uses have been deemed non

- food uses. There are currently

no established MRL s in Canada; however, there are Codex MRL s for lettuce, potato, and radish. Since there are no U.S. tolerances for tolclofos

- methyl, there are no issues of compatibility with respect to the Codex MRL s.Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 3 of 18 **Regulatory Recommendations and Resid ue Chemistry Deficiencies**  HED has examined the residue chemistry database for tolclofos

- methyl
. T here are no residue chemistry

issues that would preclude granting the t olcl o fos

- methyl seed treatment as non - food uses (40 CFR Part 158) and no food or feed tolerances are required
. Table 1 shows the proposed seed treatment use of tolclofos

- methyl as a non

- food use on corn, soy bean, cucumber, lettuce and radish can be translated to the following c rop groups . The chemical structure, nomenclature and pohys icoch emical properties of tolclofos

- methyl are presented in Table 2. Table 1: Translation of T olclofos

- methyl Seed Treatment as a Nonfood Uses

**T ranslation of T**  **olclofos -**  **methyl Seed Treatment as a Nonfood Uses Crop**  **Crop Group Number Crop Group Name**  Corn* 15 Cereal grains 16 Forage, fodder and straw Soybean* 6 Legume vegetable 7 Foliage 20 Oilseeds Cucumber* 8 Fruiting vegetable (except cucurbits) 9 Cucurbit Lettuce* 4 Leafy vegetable (except Brassica vegetables) 5 Brassica cole leafy vegetables Radish** 1 Root and tuber vegetables (except potatoes) 2 Leaves of root and tuber vegetables (human food and animal feed) 3 Bulb vegetables Cotton** *application rate: 10 g ai/100 kg seed ** application rate: 50 g ai/100 kg seedTolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 4 of 18 Table 2 : Nomenclature and Phy sicochemical P roperties of Radiolabeled Tolclofos

methyl Parameter Structure/Name/Value
Compound Structure

Common Name [Phenyl

14 C]tolclofos

-methyl (Radiolabeled) Code Names V
- 10178 IUPAC Name O
- 2,6
- dichloro

- p -tolyl O,O
- dimethyl phosphorothioate CAS Name O
- (2,6

- dichloro

- 4
- methylphenyl) O,O
- dimethyl phosphorothioate CAS Registry No. 57018
- 04
- 9 Molecular Formula C 9 H 11 Cl 2 O 3 PS Molecular Weight 305.0 (Radiolabeled, Inv No. 398)

Lot No. CFQ15407 (Valent Radiochemical Inv. No. 398) The test substance received from Sumitomo Chemical Co. was purified and isotopically diluted with tolclofos

methyl T.G. to prepare Inventory No. 398A for seed treatment Specific Activity 4.51 GBq/mmol or 887,200 dpm/μg (Inv. No. 398) 1.23 MBq/mg or 73,500 dpm/μg (Inv. No. 398A) Radiochemical Purity 98.6% (390A) Position of Radiolabel Phenyl ring
n - Octanol/Water Partition Coefficient, log P OW 4.56 at 25°C Water Solubility 1.1 ppm Vapor Pressure 57 mPa at 20°C O C l

C l H 3 C P S O C H 3 O C H 3 

## *Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 5 of 18 **Residue Analytical Method** Purification of Radiolabeled Test Substance To determine the total radioactive residue (TRR), aliquots of samples were combusted in a biological oxidizer followed b y liquid scintillation counting (LSC) of the aliquot vials. The radiolabeled test substance received from Sumitomo Chemical Co. in solid form, was dissolved with acetonitrile for LSC and HPL C analy sis. The test substance was purified as described in the following procedure. Field Corn, Cotton and Soybean Batches of field corn, cotton and soy bean seeds (one batch for each of the 3 dose levels and one batch for the untreated control per crop) were coated with treated with the appropriate amount of test substance, [phenyl

14 C]tolclofos

- me thyl . The target treatment rates were 2.5, 5.0, and 10.0g/ 100kg of seeds for the field corn and soy bean , and 10, 25, 50g/ 100 kg of seed s for cotton. The actual mean treatment rates were 2.37 + 0.24, 5.01 + 0.06, and 10.19 + 0.15 g of the test substance per 100kg of field corn seeds, 10.32 + 0.21, 22.39 + 0.63, and 51.20 + 1.14g/100 kg of cotton seeds, and 2.82 + 0.09, 5.76 + 0.30, 10.33 + 0.04g/100kg of soy bean seeds (See summary Table 3a). The radiochemical stock solution was rotary evaporated to complete dry ness (water bath temp at ~ 30 °C). The flask was kept under vacuum for an additional 30 min to allow traces of tolu ene to evaporate from the sample. The test substance was dissolved in 0.5 mL of acetonitrile, and then 0.2 mL of water was added to the solution to lower the acetonitrile concentration to 71%. The solution was divided equall y into 3 portions and each portion was loaded onto a strata

- X 1g/12 mL Giga Tube column prepared earlier by passing 1 bed

- volume each of methanol and acetonitrile, followed by 4 bed

- volumes of water. The flask containing the test substance was subsequently rinsed with 0.4 mLof 50% ac etonitrile and 0.5 mLof 40% acetonitrile and the rinses were loaded onto the columns. The columns were eluted subsequentl y with step gradients of 20

– 71% acetonitrile in water. The collected column fractions were anal yz ed b y LS C and HPL C to locate the test substance band. Radish, Lettuce and Cucumber Batches of radish, lettuce and cucumber seeds were treated with appropriate amounts of the test substance, [phenyl

14 C]tolclofos

- methyl, at the target rate of 50 and 200 g/100kg of seeds for radish, and 10 g/100 kg of seeds for lettuce and cucumber. The actual mean treatment rates were 51.6 + 0.3 and 194 + 7.6 g of the test substance per 100 kg of radish seeds, and 9.91 + 0.39 and 10.23 + 1.08 g/100 kg of lettuce and cucumber seeds, respectivel y. The actual rates were 97.0
- 103.2% of the target rates (see summary Table 3b)Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 6 of 18 The radiochemical stock solution was concentrated to 1.0 mLunder a flow of nitrogen at room temperature
. The solution was loaded onto a strata

- X 33 μm 1g/12 mLcolumn prepared earlier b y passing 2 column

- volumes of acetonitrile, followed b y 4 column

volumes of water. After the test solution entered the column, two rinses of the test substance vial with 0.5 mL of acetonitrile was loaded onto the column. The columns were eluted with step gradients of 0

– 73% acetonitrile in water. The collected column fractions were anal yz ed by L SC and HPL C to locate the test substance band.
Table 3a: Total [Phenyl

14 C]Tolclofos

- Methyl Applied to Seeds and the Treatment Rates Crop Type Dose

Level Target

Rate g/100 kg Total

Applied dpm Final

Treatment dpm Actual

Treatment Rate g/100 kg CV

% % of Target Rate
Field Corn Low 2.5 3,179,562 3,130,422 2.37 10.2 95.0

Medium 5.0 6,950,225 6,603,275 5.01 1.2 100.2 High 10.0 14,204,616 13,514,016 10.19 1.5 101.9
Cotton Low 10.0 4,723,724 4,707,495 10.32 2.1 103.2

Medium 25.0 11,255,999 10,246,959 22.39 2.8 89.5 High 50.0 24,121,406 23,153,086 51.20 2.2 102.4 Soybean Low 2.5 1,644,133 1,627,878 2.82 3.2 112.8

Medium 5.0 3,305,600 3,284,245 5.76 5.3 115.3 High 10.0 6,139,779 5,919,522 10.33 0.4 103.3
Table 3b : Total [Phenyl

14 C]Tolclofos

- Methyl Applied to Seeds and the Treatment Rates Crop Type Target

Rate g/100 kg Analyzed

Subsample No. Applied

dpm/g of seeds Mean

Applied dpm/g of seeds Applied

Treatment Rate g/100 kg CV

% % of Target Rate

Radish 50 1 38,176,246 37,926,566 51.6 0.7 103.2 2 37,668,537 3 37,934,915
200 1 142,696,560 142,616,028 194.0 3.9 97.0

2 136,965,195 3 148,186,329
Lettuce 10 1 7,054,295 7,280,646 9.91 4.0 99.1 2 7,180,130 3 7,607,512 Cucumber 10 1 8,010,039 7,516,276 10.23 10.6 102.3Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 7 of 18 2 7,939,973 3 6,598,815 [cv=coefficient of variation; dpm=disintegrations per minute]
HED Conclusions An adequate data collection method is available for tolclofos

- methyl residues in/on crops, liquid scintillation counting (LSC) and HPL C. Batches of s eeds were treated with appropriate amounts of the test substance, [phenyl

14 C]tolclofos

- methyl. The target treatment rates were 2.5, 5.0 and 10.0 g/100 kg of seeds for the field corn , cotton and soy bean, and 10, 50 and 20 0 g/100 kg of seeds for radish, lettuce , and cucumber
. The percentage of target rate ranged from 89.5

- 115.3. **Storage Stability** Crop samples were collected at their proper growth stage. The treated batches of seeds were placed in labeled 30 or 50 mLglass vials fitted with caps and shipped at ambient temperature to Research f or Hire via commercial carriers on a next - day delivery schedule. All RAC samples were shipped frozen on Dry I ce from Research f

or Hire b y commercial carriers on overnight delivery schedules to Valent Technical Center in Dublin CA
. The samples were anal yz ed within 30 day s with the exception of soy bean forage (stored for 41 days ) ( Table 4 )
. Additionally , samples of radish tops were stored for up to 13.7 months before extraction (Table 5)
. Table 4 : Harvest and TRR Analy sis Dates of Radish, Cucumber and Lettuce Samples
Sample Description Date

Harvested Date

Shipped Date

Processe d Date of

TRR analysis Storage

period (days) before TRR analysis Radish 07/10/09 07/13/09 07/20/09 07/28/09 18
Cucumber 08/10/09 08/12/09 8/18/09 08/31/09 21
Lettuce 07/22/09 08/04/09 8/12/09 08/19/09 28 Soybean forage 07/30/07 07/31/07 09/04/07 09/10/07 41 Soybean seed and hay 09/21/07 09/25/07 10/04/07 10/11/07 16Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 8 of 18 Field corn forage 08/17/07 08/21/07 08/28/07 09/07/07 17 Field corn grain and stover 09/14/07 09/17/07 09/21/07 09/25

- 26/07 8
- 9
Cotton seed 10/18/07 10/23/07 11/01/07 11/12/07 20 Cotton gin trash 10/18/07 10/23/07 11/06/07 11/14/07 22

Table 5 : Harvest an d TRR Analy sis Dates of Radish Top Samples
Sample Description Date Harvested and

Storage Started Date Extracted

for Analysis Storage period

(days) before extraction analysis Treated Radish Tops 07/10/09 08/24/10 410 (13.5 months) Spiked Control Radish Tops 07/10/09 08/30/10 416 (13.7 months) To determine the stability of radish tops under frozen storage, the registrant also submitted a freezer storage stability study for tolclofos

- methyl in/on lettuce ( MRID 201000183 , 3/14/02). The study examined the stability

of tolclofos

- methyl fortified at 0.1 mg/kg in lettuce stored frozen at < - 18 o C for 0, 3, 6, 12 and 18 months
. The overall percent recovery

of the method for lettuce freshly fortified with 0.1 mg/kg of tolclofos

methyl ranged from 83

- 102%. Lettuce were extracted with acetone: water (2:1 v:v). For liquid

- liquid partition ethyl acetate/ cyclohexane and sodium chloride was added and after repeatedly mixing excess water was separated. The residue containing fraction is concentrated and anal yz ed for residues of tolclofos

- methyl by gas chromatography

using a flame photometric detector (FPD, phosphorus mode). The LOQ was 0.01 ppm. HED Conclusions The available storage stability data for soy

bean, corn, cotton, cu cumber, lettuce , and radish is adequate. The dates that the samples were planted , collected and analyzed were less than 30 day s except for so ybean forage which was stored for 41 days . The free zer storage period before the extraction analy sis of treated radish tops was 13.7 months. The petitioner submitted the results of storage stability for lettuce that showed tolclofos

methyl was stable for up to 18 months.Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 9 of 18 **Radiolabeled Field Trials** F ield corn, cotton and soy bean seeds were planted in outdoor field plots (Greenville, MS) following normal agricultural practices. Each plot was approximately 30 × 30 inches and had one row of plants grown from treated seeds of one dose level at 3 inches ap art from each other. The boundary of each plot was surrounded by a plastic edge liner buried in soil with a minimum of about 6 inches below ground. Five seeds from each dose level were planted per plot, and 3 plants of normal crop density were selected f

or sampling to provide triplicate samples for each RAC. Radish, lettuce and cucumber seeds were also planted in outdoor field plots (Stockton, CA) following the normal agricultural practices. Each plot was approximately 2 ft × 2 ft, in which seeds were planted at appropriate crop density for the respective plant. The boundary of each plot was surrounded b y a plastic

- lined wooden frame, which was buried in soil about 6 inches below ground. Duplicate plots were planted with treated seeds from each dose level and approximately 30 seeds were planted per plot for radish, 20 seeds per plot for lettuce and 10 seeds per plot for cucumber. A total of 5 plots (1 for untreated control, 2 for low dose rate and 2 for high dose rate) were used for the radish crop, and 3 plots (1 control and 2 treated) were planted with each of the lettuce and cucumber crops. **Treatment of Field Corn Seed**  Field corn forage, stover and grain were harvested from the untreated control plot and three treatment plots which were p lanted with seeds treated with 14 C
- tolclofos methyl at the nominal dose rates of 2.5, 5.0 , and 10.0 g/100 kg of seeds. Approximately

0.2 mg, 0.3 mg and 0.6 mg of Rizolex 50FLFormulation were transferred to 3 separate 25 mL flask s marked for low, me dium and high dose, respectively . After adding 43 μL of water to each flask, the formulation blank and water were mixed by sonication. The batches of 15 field corn seeds were then transferred to the respective flask and swirled gently

and briefl y to coat them evenl y with the formulation blank. The seeds were then transferred onto labeled weighing

- tray s and allowed to dry at room temperature. Each of the 15 seeds of the low dose batch was individually spiked with 1.0 μLof the test substance stock soluti on 390A and 1.0 μLof the dilution solution 390AD1. Each of the 15 seeds of the medium dose batch was individually spiked with 3.0 μLof 390A only . Finally , each of the 15 seeds of the high dose batch was individually spiked with 6.0 μLof 390A and 1.0 μ L of the dilution solution 390AD3. I dentical triplicate aliquots of the stock and dilution solution used in each batch as the dose were transferred to empty vials, diluted to 5.0 mL

with acetonitrile, and subaliquots of these dilution solutions were count ed by L SC for dose verification. Three treated seeds from each batch were rinsed individually with 5.0 mL of acetonitrile. The radioactivity applied to each seed was verified by LSC anal ys is of the rinse and combustion anal ysis of the rinsedTolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 10 of 18 seed. The remaining 12 treated seeds were allowed to dry at room temperature, then placed individually

in labeled glass vials and shipped to Research f or Hire for planting. All of the corn forage, stover, and grain samples showed TRR levels below the L OQ (0.0003 ppm), and the majority of the corn samples had radioactivity level below the limit of detection (0.0001 ppm). The total radioactive residues in field corn are presented in Table 6. **Treatment of Cotton Seeds** Cotton seed and gin trash were harvested from the untreated control plot and three treatment plots which were planted with seeds treated with 14 C
- tolclofos methyl at the nominal dose rates of 10, 25, and 50 g/100 kg of seeds. Approximately

0.2 mg, 0.5 mg and 1.1 mg of Rizolex 50FLformulation b lank were transferred to 3 separate 25 mLcapacity flasks marked for low, medium and high dose, respectivel y. After adding 28 μL of water to each flask, the formulation blank and water were mixed by

sonication. The batches of 15 cotton seeds were then transferred to the respective flask and swirled gently and briefly

to coat them evenl y with the formulation blank. The seeds were then transferred onto labeled weighing

- tray s and allowed to dry at room temperature. Each of the 15 seeds of the low dose batch was individually spiked with 2.0 μLof the test substance stock solution 390A and 1.0 μLof the dilution solution 390AD2. Each of the 15 seeds of the medium dose batch was individually spiked with 5.0 μLof 390A and 1.0 μLof 390AD3. Finall y, each of the 15 s eeds of the high dose batch was individually

spiked with 10.0 μLof 390A and 1.0 μLof 390AD4. I dentical triplicate aliquots of the stock and dilution solution used in each batch as the dose were transferred to empty vials, diluted to 5.0 mL with acetonitrile, and subaliquots of these dilution solutions were counted by L SC for dose verification. Three treated seeds from each batch were rinsed individually with 5.0 mLof acetonitrile. The radioactivity applied to each seed was verified by L SC anal ys is of the rinse and combustion analy sis of the rinsed seed. The remaining 12 treated seeds were allowed to dry at room temperature, then placed individually

in labeled glass vials and shipped to Research f or Hire for planting. All of the cotton seed meal, seed hull and gin trash samples (except one replicate of high dose gin trash sample) showed TRR levels below the LOQ (0.00035 ppm), and the majority of the cotton samples had radioactivity level below the limit of detection (0.0001 ppm). The total radioactive residues in cotton are presented in Table 7. **Treatment of Soybean Seeds** Soy bean forage, hay

and seed were harvested from the untreated control plot and three treatment plots planted with seeds treated with 14 C
- tolclofos methyl at the nominal dose rates 2.5, 5.0 , and 10 g/100 kg of seeds. Approximately 0.1 mg and 0.3 mg of Rizolex 50FL formulation b lank were transferred to 3 separate 25 mLcapacity flasks marked for low, medium and high dose, respectivel y.Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 11 of 18 After adding 10 μL of water to each flask, the formulation blank and water were mixed by sonication. The batches of 15 soy bean seeds were then transferred to the respective flask and swirled gently

and briefl y to coat them evenl y with the formulation blank. The seeds were then transferred onto labeled weighing

- tray s and allowed to dry at room temperature. Each of the 15 seeds of the low dose batch was individually spiked with 2.0 μLof the dilution solution 390AD5. Each of the 15 seeds of the medium dose batch was individually

spiked with 1.0 μLof the test substance stock solution 390A and 1.0 μLof 390AD5. Finall y, each of the 15 seeds of the high dose batch was individually spiked with 2.0 μLof 390A and 2.0 μLof 390AD5. Identical triplicate aliquot s of the stock and dilution solution used in each batch as the dose were transferred to empty vials, diluted to 5.0 mLwith acetonitrile, and subaliquots of these dilution solutions were counted by L SC for dose verification. Three treated seeds from each batch were rinsed individually with 5.0 mLof acetonitrile. The radioactivity applied to each seed was verified by L SC anal ys is of the rinse and combustion analy sis of the rinsed seed. The remaining 12 treated seeds were allowed to dry at room temperature, then placed individuall y in labeled glass vials and shipped to Research f or Hire for planting. Three additional batches of soy bean seeds (10 in each batch) were treated for replanting after some so yb ean seeds from the first treatment failed to germinate (the germination rate of so ybean seeds was generall y low in all dosing levels including the untreated control). The repeat treatment procedure was similar to the one described above, except that the treated seeds were not anal yz ed in addition to the anal ys is of the dosing aliquots. All of the soy

bean forage and seed samples showed TRR level below the LOQ (0.00034 ppm), and the majority of them had radioactivity level below the limit of detection (0.0001 ppm). The low and high dose so ybean hay samples show ed mean TRR levels of 0.00049ppm and 0.00067, respectivel y. The total radioactive residues in soybean are presented in Table 8. **Treatment of Radish Seed**  Two batches (5.0 g each) of radish seeds were weighed into separate containers. Into a 30 mLcapac ity

glass vial, 216 μLof the test substance solution in acetonitrile, 398A, was transferred. The solution was evaporated to dry ness under nitrogen at room temperature, and the residue was redissolved in 50 μLof acetonitrile. An aliquot of 3.59 μLof th e formulation blank and 77 μLof water were added to the radiolabeled solution in the vial. The first 5.0 g batch of the radish seeds was transferred to the vial. The seeds were mixed with the slurry

by gentle shaking until no liquid was visible in the vi al. The seeds were poured out of the vial and discarded. New slurry was prepared in the emptied vial as described above, using 216 μLof 398A (concentrated to 50 μL), 3.59 μL of formulation blank, and 77 μLof water. The slurry was mixed well using the v ortex mixer, and spread around the inner wall of the vial. The second 5.0 g batch of the radish seeds was transferred to the vial. The seeds were mixed with the slurry by gentle shaking until no liquid was visible in the vial. Three subsamples ( 0.25 g ea ch) of these treated seeds were withdrawn from the vial for anal ys is. Seeds in each subsample were rinsed with 2 lots of acetonitrile (5 mL each), and the pooled seed rinse (total volume, 10 mL)Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 12 of 18 was anal yz ed b y LSC. The rinsed seeds were air

- dried at roo m temperature before combustion analy sis. A dose rate of 200 g/100kg of radish was also anal yz ed. Only the radish root samples grown from seeds treated at the rate of 51.6 g/100 kg showed TRR level below the L OQ. All other radish samples showed residues a bove 0.005 ppm. The radish tops grown from seeds treated at the rates of 51.6 g/100 kg and 194.0 g/100 kg contained 0.02

- 0.03 ppm and 0.04

- 0.06 ppm, respectively. The radish root from the higher dose rateshowed residue levels of ~0.03 ppm. The total radioactive residues in radish are presented in Table 9. **Treatment of Lettuce Seeds** Two batches (5.0 g each) of lettuce seeds were weighed into separate containers. Into a 30 mLcapacity glass vial, 0.72 μLof the formulation blank, 43 μLof the test sub stance solution in acetonitrile, 398A, and 87 μLof water were transferred. The slurry was mixed well using a vortex mixer, and spread around the inner wall of the vial. The first 5.0 g batch of the lettuce seeds was transferred to the vial and the seeds were mixed with the slurry by gentle shaking until no liquid was visible in the vial. The seeds were poured out of the vial and discarded. New slurry was prepared in the emptied vial as described above, using 0.72 μLof formulation blank, 43 μLof 398A, and 87 μL of water. The slurry was mixed well using the vortex mixer, and spread around the inner wall of the vial. The second 5.0 g batch of the lettuce seeds was transferred into the vial and the seeds were mixed with the slurry by gentle shaking until n o liquid was visible in the vial. Three subsamples (ca 0.25 g each) of these treated seeds (designated as LT) were withdrawn from the vial for anal ys is. Seeds in each subsample were rinsed with 2 lots of acetonitrile (5 mL each), and the pooled seed rins e (total volume, 10 mL) was anal yz ed by L SC. The rinsed seeds were air

- dried at room temperature before combustion anal ys is. The lettuce samples grown from seeds treated at the rate of 9.91 g/100 kg showed TRR level below the minimum quantification limit . The control lettuce samples also showed no radioactivity above background. The total radioactive residues in lettuce are presented in Table 9.
**Treatment of Cucumber Seeds** Two batches (10 g each) of cucumber seeds were weighed into separate containers . Into a 50 mLcapacity glass vial, 1.43 μLof the formulation blank, 86 μLof the test substance solution in acetonitrile, 398A, and 173 μLof water were transferred. The slurry was mixed well using a vortex mixer, and spread around the inner wall of the vial. The first 10 g batch of the cucumber seeds was transferred to the vial and the seeds were mixed with the slurry

by gentle shaking until no liquid was visible in the vial. The seeds were poured out of the vial and discarded. New slurry was prepared in the emptied vial as described above, using 1.43 μLof formulation blank, 86 μLof 398A, and 173 μLof water. The slurry

was mixed well using the vortex mixer, and spread around the inner wall of the vial. The second 10 g batch of the cucumber seeds was transferred into the vial and the seeds were mixed with the slurry by gentle shaking until no liquid wasTolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 13 of 18 visible in the vial. Three subsamples (ca 0.25 g each) of these treated seeds (designated as CT) were withdrawn from the vial for analy sis. Seeds in each subsample were rinsed with 2 lots of acetonitrile (5 mL each), and the pooled seed rinse (total volume, 10 mL) was anal yz ed b y LSC. The rinsed seeds were air

- dried at room temperature before combustion analy sis. The total radioactive residues in fie ld corn are presented in Table 6. The cucumber samples grown from seeds treated at the rate of 10.23 g/100 kg showed TRR level below the minimum quantification limit. The control cucumber samples also showed no radioactivity above background. The total radioactive residues in lettuce are presented in Table 9.
Table 6 : Total Radioactive Residue in Field Corn RAC Samples Sample Description Dose level

[g/100 kg] Sample

No. Rep. No. Mean Aliquot wt., g Mean Aliquot dpm TRR

Mean dpm/g ppm

μg/g

Field Corn Forage Control [0.0] 62 1 0.266 11 <126 <0.00030

63 2 0.262 5 <35 <0.0000 8 64 3 0.274 4 <34 <0.00008 Low [2.5] 65 1 0.278 10 <121 <0.00028

66 2 0.264 4 <35 <0.0000 8 67 3 0.258 4 <36 <0.0000 8 Medium [5.0] 68 1 0.261 17 <129 <0.00030

69 2 0.262 3 <35 <0.0000 8 70 3 0.270 8 <34 <0.00008 High [10.0] 71 1 0.260 12 <129 <0.00030

72 2 0.266 3 <35 <0.00008 73 3 0.265 5 <35 <0.00008

Field Corn Stover Control [0.0] 74 1 0.283 10 <116 <0.00027

75 2 0.259 4 <35 < 0.00008 76 3 0.251 1 <36 <0.00008 Low [2.5] 77 1 0.256 11 <129 <0.00030

78 2 0.247 3 <37 <0.00008 79 3 0.258 5 <35 <0.00008 Medium [5.0] 80 1 0.260 12 <127 <0.00030

81 2 0.262 4 <34 <0.00008 82 3 0.249 5 <36 <0.00009 High [10.0] 83 1 0.253 11 <130 <0.00030

84 2 0.259 6 <35 <0.00008 85 3 0.267 7 <34 <0.00008

Field Corn Grain Control

[0.0] 86 1 0.286 10 <119 <0.000 0 8

87 2 0.295 5 <32 <0.000 27 88 3 0.284 2 <33 <0.00007 Low [2.5] 89 1 0.283 13 <120 <0.000 28

90 2 0.276 6 < 34 <0.00007 91 3 0.289 6 <32 <0.00008 Medium 92 1 0.293 21 <116 <0.00027Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 14 of 18 [5.0] 93 2 0.279 25 <122 <0.00028

94 3 0.280 21 <121 <0.00028 High [10.0] 95 1 0.292 5 <32 <0.00007

96 2 0.284 3 <33 <0.00008 97 3 0.300 3 <31 <0.00007
Table 7 : Total Radioactive Residue in Cotton RAC Samples Sample Description Dose level

[g/100 kg] Sample

No. Rep. No. Mean Aliquot wt., g Mean Aliquot dpm TRR

Mean dpm/g ppm

μg/g

Cotton Seed Meal Control

[0.0] 38P 1 0.248 2 <41 <0.00009

39P 2 0.255 5 <39 <0.00009 40P 3 0.247 3 <41 <0.00009 Low [10.0] 41P 1 0.254 1 <40 <0.00009

42P 2 0.264 3 <38 <0.00009 43P 3 0.246 0 <41 <0.00010 Medium [25.0] 44P 1 0.254 8 <40 <0.00009

45P 2 0.248 2 <48 <0.00009 46P 3 0.264 1 <38 <0.00009 High [50.0] 47P 1 0.259 7 <39 <0.00009

48P 2 0.251 4 <40 <0.00009 49P 3 0.260 5 <39 <0.00009

Cotton Seed Hull Control

[0.0] 38H 1 0.257 2 <36 <0.00008

39H 2 0.247 2 <38 <0.00009 40H 3 0.243 19 <139 <0.00033 Low [10.0] 41H 1 0.257 4 <36 <0.00008

42H 2 0.248 3 <38 <0.00009 43H 3 0.249 2 <37 <0.00009 Medium [25.0] 44H 1 0.235 7 <40 <0.00009

45H 2 0.248 4 <38 <0.00009 46H 3 0.244 3 <38 <0.00009 High [50.0] 47H 1 0.255 7 <37 <0.00009

48H 2 0.257 6 <36 <0.00008 49H 3 0.253 5 <37 < 0.00009

Cotton Gin Trash Control

[0.0] 50 1 0.246 1 <40 <0.00009

51 2 0.244 0 <41 <0.00010 52 3 0.255 1 <39 <0.00009 Low [10.0] 53 1 0.263 8 <38 <0.00009

54 2 0.263 2 <38 <0.00009 55 3 0.255 4 <39 <0.00009 Medium [25.0] 56 1 0.259 13 <138 <0.00032

57 2 0.267 8 <37 <0.00009 58 3 0.251 2 <40 <0.00009 High [50.0] 59 1 0.239 25 <150 <0.00035

60 2 0.251 21 <142 <0.00033 61 3 0.248 37 151 0.00035Tolclofos

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and Residue Data D395999

Page 15 of 18 Table 8 : Total Radioactive Residue in Soy bean RAC Samples Sample Description Dose level

[g/100 kg] Sample

No. Rep. No. Mean Aliquot wt., g Mean Aliquot dpm TRR

Mean dpm/g ppm

μg/g

Soybean Forage Control

[0.0] 2 1 0.266 13 <129 <0.00030

3 2 0.254 1 <37 <0.0000 9 4 3 0.277 1 <34 <0.0000 8 Low [2.5] 5 1 0.268 12 <127 < 0.00030

6 2 0.268 5 <35 <0.00008 7 3 0.263 2 <36 <0.0000 8 Medium [5.0] 8 1 0.268 14 <127 <0.00030

9 2 0.269 7 <35 <0.00008 10 3 0.266 4 <35 <0.00008 High [10.0] 11 1 0.249 15 <137 <0.00032

12 2 0.249 5 <38 <0.00009 13 3 0.265 7 <36 < 0.00008

Soybean Hay Control

[0.0] 14 1 0.222 15 <151 <0.00035

15 2 0.209 12 <161 <0.00038 16 3 0.195 13 <172 <0.00040 Low [2.5] 17 1 0.221 43 194 0.00045

18 2 0.206 50 200 0.00047 19 3 0.212 42 162 < 0.00038 Medium [5.0] 20 1 0.209 32 <160 <0.00038

21 2 0.200 27 <168 <0.00039 22 3 0.209 23 <160 <0.00038 High [10.0] 23 1 0.209 40 188 0.00044

24 2 0.198 35 176 0.00041 25 3 0.194 97 497 0.00116

Soybean Seed Control

[0.0] 26 1 0.255 5 <39 <0.000 09

27 2 0.263 0 <38 <0.00009 28 3 0.261 0 <38 <0.00009 Low [2.5] 29 1 0.260 7 <38 <0.00009

30 2 0.247 2 <40 <0.00009 31 3 0.254 2 <39 <0.00009 Medium [5.0] 32 1 0.244 7 <41 <0.00010

33 2 0.252 3 <39 <0.00009 34 3 0.249 3 <40 <0.000 09 High [10.0] 35 1 0.258 11 <138 < 0.00032

36 2 0.258 10 <139 <0.00032 37 3 0.249 13 <143 <0.00034

Table 9 : Total Radioactive Residue Levels in Radish, L ettuce and Cucumber Samples Grown from Seeds Treated with [Phenyl

14 C]Tolclofos

- MethylTolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 16 of 18 Sample Description Dose Rate

g/100 kg Plot

ID Mean Aliquot wt., g Mean Aliquot dpm CV% TRR

Mean dpm/g ppm

μg/g
Radish Tops 0.0 CR1 0.2387 1 nm <245 <0.003

51.6 TR1 0.2384 508 34.7 a 2,133 0.029

TR2 0.2415 344 36.6 b 1,435 0.020 194.0 TR3 0.2447 1,140 8.9 4,655 0.063

TR4 0.2439 682 7.3 2,796 0.038

Radish Roots 0.0 CR1 0.2427 0 nm <241 <0.003

51.6 TR1 0.2362 61 nm <250 <0.003

TR2 0.2401 32 nm <243 <0.003 194.0 TR3 0.2407 563 17.1 b 2,343 0.032

TR4 0.2401 560 11.5 b 2,331 0.032
Lettuce 0.0 CL1 0.2375 0 nm <226 < 0.003

9.9 TL2 0.2406 2 nm <223 <0.003

TL3 0.2442 0 nm <220 <0.003 Cucumber 0.0 CF1 0.2542 0 nm <211 <0.003

10.2 TF1 0.2555 0 nm <210 <0.003

TF2 0.2535 0 nm <212 <0.003 [a: mean of 15 aliquots; b: mean of 9 aliquots, all others: mean of 3

– 6 aliquots] **Characterization of Radish Seeds** Since the radish tops grown for seeds treated at the rate of 51.6 g/100 kg, showed residues above 0.005 ppm, additional characterization was provided. Two treated and one control crop samples were extracted and anal yzed. The treated samples were radish tops (TRR = 0.029 ppm and TRR = 0.020 ppm) from replicate plots TR1 and TR2, respectivel y. The control radish tops grown from untreated seeds were spike d with radiolabeled tolclofos

- methyl and anal yz ed to determine the efficiency of the extraction method. To investigate the nature of these low level residues further and to quantify the test substance, tolclofos

- methyl w as extracted with aqueous acetonitrile, which was separated into acetonitrile and aqueous extracts. The replicate samples showed similar total radioactive residue (TRR) distribution in the extracts and in the post

- extraction solids (PES). Approximately

81 and 80% of TRR (0.023 and 0.016 pp m) was extractable from samples TR1 and TR2 , respectivel y, while about 19 and 20% (0.005 and 0.004 ppm) remained bound to PES. The major portion of the extractable residue (60% TRR, 0.017 ppm in TR1, and 56% of TRR, 0.011 ppm in TR2) was in the aqueous extract. Approximately

22% of TRR (0.006 ppm) in TR1 and 24% (0.005 ppm) in TR2 remained soluble in the acetonitrile extract. The extraction TRR levels in TR1 and TR2,Tolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 17 of 18 respectivel y, were 96 and 102% of the levels determined by combustion analy sis in the orig inal study . Extraction of approximately 100 g of untreated control radish tops spiked with [ 14 C]tolclofos

- methyl under similar conditions showed that the entire spiked radioactivity

was extractable (97% TRR in acetonitrile extract, and 3% in aqueous extrac t). The acetonitrile extracts were purified using strataX column chromatography , and finally anal yz ed b y HPL C. Both acetonitrile and aqueous extracts of treated radish tops samples, TR1 and TR2, showed multiple components of polar low level radioactive residues, but did not contain any detectable amount of tolclofos

- methyl. Acetonitrile extract of control radish tops spiked with [ 14 C]tolclofos

- methyl, which was subjected to identical purification procedure, showed a single peak of [ 14 C]tolclofos

- methyl in the HPL C anal ys is. The extraction and HPL C results are presented in Table 10
. Table 10 : Extraction and HPL C Results for Tolclofos

- methyl in Radish Tops
Extract/Residue Fraction Radish Tops Sample Spiked Control Treated TR1 Treated TR2 % TRR ppm % TRR ppm % TRR ppm Acetonitrile Extract Tolclofos

- methyl 97.3 0.027 ND ND ND ND Unknowns (multiple components) ND ND 21.7 0.006 23.8 0.005 Aqueous Extract Unknowns (multiple components) 2.7 <0.001 59.5 0.017 56.2 0.011 Total extractable 100.0 0.027 81.2 0.023 80.0 0.016 Total bound in PES 0.0 0.0 18.8 0.005 20.0 0.004 Total radioactive residue 100.0 0.027 100.0 0.028 100.0 0.020 [ND= not detected]
HED Conclusions Batches of radish, lettuce , cucumber, corn, so ybean , and radish seeds were treated with formulated [ phenyl

14 C]tolclofos

- methyl
. The target treatment rates were 50 g/100 kg of seeds for cotton and radish, and 10 g/100 kg of seeds for corn, so yb ean, lettuce and cucumber. The results showed that the seeds treated with [phenyl

14 C]tolclofos

- methyl did not take up the radiolabeled test substance residue (<5 ppb). Only the radish top samples grown from seeds treated at the rate of 51.6 g/100 kg showed TRR level residues above 0.005 ppm. The radish tops grown from seeds c ontained residues at 0.02

– 0.03 ppm. Since the radish tops grown for seeds showed resid u es above 0.005 ppm, additionalTolclofos

- methyl Summary of Anal ytical Chemistry

and Residue Data D395999

Page 18 of 18 characterization of the residues was provided. Two treated and one control crop samples were extracted and anal yzed. The treated samp les were radish tops (TRR = 0.029 ppm and TRR = 0.020 ppm) from replicate plots TR1 and TR2, respectivel y. The control radish tops grown from untreated seeds were spiked with radiolabeled tolclofos

- methyl and anal yz ed to determine the efficiency of the extraction method. The radish tops grown for seeds were extracted and anal yz ed b y HP LC . Both acetonitrile and aqueous extracts showed some polar radioactive residues, but did not contain any detectable amount of tolclofos

- methyl. **REFERENCES** DP#: 362358 Subject: Tolclofos

- methyl: Non

- food Use for Seed Treatment From: S. Piper To: C. Giles

- Parker Date: 02/28/09 MRIDs: NA