Absorption, Distribution and Excretion
This study investigated the metabolism of 14C-pyrrole-labeled flonicamid in goats… Two goats were orally fed radiolabeled flonicamid for four consecutive days at a concentration equivalent to 100 ppm in their feed. On day 4, the radioactive residues, calculated as flonicamid, were: tenderloin 0.07 mg/kg, fat 0.19 mg/kg, liver 5.8 mg/kg, kidney 2.9 mg/kg, and milk 2.2 mg/kg. After organic solvent extraction, 35% of the radioactive residue was released from the liver, 76% from the muscle, 50% from the kidney, 35% from the fat, 87% from the milk, and 90% from the milk. Treatment of the solid residues after solvent extraction of the liver, kidney, and muscle with proteases released 75-91% of the remaining radioactivity. After derivatization with 2,4-dinitrofluorobenzene, less than half of the released radioactive material was identified as protein.
For eight consecutive days, five laying hens were fed gelatin capsules containing [(14)C-pyrrole]flufenican, with a feed addition equivalent to approximately 89 ppm. The vast majority of the radiolabeled residues were excreted in feces (88-102% of the total dose). The levels of radioactive residues in tissues and eggs, calculated as flufenican, were as follows: liver, 8.9 mg/kg; muscle, 0.12 mg/kg; fatty skin, 0.25 mg/kg; peritoneal fat, 0.17 mg/kg; egg yolk, 1.8 mg/kg (day 7); egg white, 0.054 mg/kg (day 7). A series of organic solvent extraction experiments showed that 61%, 33%, 62%, 42%, 74%, and 83% of the radioactivity (TRR) were released from the liver, kidney, muscle, fatty skin, egg white, and egg yolk, respectively. The remaining solids after solvent extraction of liver (33% TRR), kidney (54%), and muscle (34%) were dissolved with a protease and characterized with 2,4-dinitrofluorobenzene. The protease dissolved 54% of the unextracted radioactivity in the liver, 63% in the kidney, and 67% in the muscle. Approximately 25% of the released radioactivity (<10% TRR) was derivatized by 2,4-dinitrofluorobenzene at pH 2, indicating the presence of amino groups at the amino terminus. Alkaline hydrolysis (15% KOH, 95°C) released all remaining radioactive material (33% TRR) from the solvent-extracted liver, but these radioactive materials could only be identified as acidic polar compounds. Approximately 69%, 24%, 14%, 44%, and 29% TRR were identified in eggs, liver, kidneys, muscle, and fatty skin, respectively…
In a feeding trial, three groups of dairy cows (three cows per group) were fed diets supplemented with 0.55 ppm, 1.6 ppm, or 5.5 ppm flonicamid for 28–30 days. The residues of flonicamid and its metabolites (determined by CGA-192155 (2,2-difluorobenzo[1,1]dioxane-4-carboxylic acid)) were only detectable at the highest feeding level (5.5 ppm)…In ruminant tissues, no quantifiable residues were detected at feeding levels of 60 times (dairy cows) and 80 times (beef cows) of the calculated dietary load. At a feeding level of 5.5 ppm, flupyradifurone and its metabolites were detected in the liver and kidneys at concentrations of 0.014–0.017 mg/kg and 0.022–0.025 mg/kg, respectively. No flupyradifurone was detected in fat or muscle. The transdermal absorption rate of flupyradifurone (excluding substances bound to the skin) was low in vivo in rats (< 5%) and also low in vitro in human skin (< 0.5%). In an in vitro rat skin penetration study, skin absorption at low doses was comparable to in vivo results (< 2%), but at high doses, skin absorption significantly overestimated in vivo absorption (38%). Metabolites/Metabolites: Two goats were orally administered radiolabeled flupyradifurone for four consecutive days at doses equivalent to 100 ppm in their feed… The major component identified in muscle was flupyradifurone, accounting for 24% and 43% of the total residues in both goats, respectively. Similarly, flonicamid was also a major component of omental fat residues, accounting for 83% of the total residues. The major metabolites identified in muscle were sulfate conjugates of flonicamid 2-hydroxy or 5-hydroxy derivatives (accounting for 22% and 2% of the total residues, respectively). Minor metabolites identified in muscle (< 10% TRR) included flonicamid 2-O-glucuronide and 5-O-glucuronide derivatives. (Location numbers refer to the pyrrole ring.) Approximately 50% of the residues in muscle and 83% of the residues in fat were identified. Multiple components were found in the kidneys and liver. Components identified in the kidneys included: flonicamid 2-O-glucuronide derivatives (23% TRR); 7'-O-glucuronide derivatives (8% TRR); 5-O-glucuronide derivatives (15% TRR); flonicamid (2% TRR); and 2- or 5-O-sulfate esters (0.7% TRR), with an overall identification rate of 48%. In the liver, only flonicamid (14% TRR) was identified. Two unstable compounds were also found (24% TRR). No compounds without a pyrrole-phenyl linkage were identified. Five laying hens were fed gelatin capsules containing [(14)C-pyrrole]fluonicamid for eight consecutive days, at a rate equivalent to approximately 89 ppm of flonicamid in the feed… The major metabolites identified in eggs were sulfate conjugates of flonicamid 1-hydroxy derivatives (40% TRR), succinic acid derivatives (10% TRR), and sulfate conjugates of 2-hydroxy or 5-hydroxy derivatives (13% TRR). Fluonicamid was present in low concentrations in eggs (2.1% TRR). In the liver, only the significant metabolite succinic acid derivative was identified, at approximately 6% TRR. Metabolites identified in the kidneys were glucuronide conjugates of 2-hydroxy or 5-hydroxy derivatives (4.7% TRR), flonicamid (2.6% TRR), and a 7'-hydroxy derivative (2.8% TRR). In the pectoral muscles, the main components identified were flonicamid (29% TRR) and sulfate conjugates of a 1-hydroxy derivative. Similar findings were observed in the skin with attached fat, containing flonicamid (9.8%) and sulfate conjugates of a 1-hydroxy derivative (14%). Based on characterization and identification of hen metabolism, it was concluded that poultry metabolism involves oxidation at the C-2, C-5, and N-1 positions of the pyrrole ring and at the C-7' position of the benzodioxane ring. This subsequently forms sulfate or glucuronide conjugates. The C-2 hydroxypyrrole is further oxidized to 2,5-dioxo-2,5-dihydropyrrole and succinic acid derivatives. The latter two compounds are specific to poultry. The remaining metabolites found in hens, as well as all metabolites found in ruminants, were also found in rats. Flupyradifurone and its metabolite (identified as 2,2-difluoro-1,3-benzodioxane-4-carboxylic acid) are stable for at least 12 months in frozen muscle and at least 18 months in frozen liver, milk, and eggs. Following oral administration of radiolabeled flupyradifurone, the radiolabeled substance is rapidly and extensively absorbed (approximately 80% of the administered dose), widely distributed, extensively metabolized, and rapidly excreted, primarily in feces (approximately 80%), bile (approximately 70%), and a small amount in urine (approximately 20%). Peak plasma concentrations are reached within 1 hour of administration. Flupyradifurone elimination is biphasic, with a first-phase half-life of 2 to 5 hours and a second-phase half-life of 30 to 60 hours. Flupyradifurone is rapidly cleared from blood and tissues, thus accumulating very little. The metabolism of flonicamid primarily occurs through the oxidation of the pyrrole ring, producing a major (57% of the administered dose) and a minor (4% of the administered dose) oxopyrrole metabolite. Hydroxylation of the benzene ring generates the corresponding phenolic metabolite, accounting for 2% of the administered dose. These phase I metabolites are subsequently excreted as glucuronic acid and sulfate conjugates, comprising approximately 75% of the administered dose along with unabsorbed and unmetabolized flonicamid. Dimerization of the hydroxypyrrole metabolite produces a dark blue metabolite. In a feeding trial, three groups of dairy cows (three cows per group) were fed diets supplemented with 0.55 ppm, 1.6 ppm, or 5.5 ppm flonicamid for 28–30 days. Residues of flonicamid and its metabolites (determined by CGA-192155 (2,2-difluorobenzo[1,1]dioxane-4-carboxylic acid)) were quantifiable only at the highest feeding level (5.5 ppm)... Tissue samples from dairy cows fed a 5.5 ppm diet were analyzed only. Residues of flonicamid and its metabolites were not detected. The limit of quantitation (LOQ) in muscle was 0.01 mg/kg, and in liver, kidney, and adipose tissue (perirhinoid and omentum) it was 0.05 mg/kg. Organic nitriles are converted to cyanide ions in the liver by cytochrome P450 enzymes. Cyanide is rapidly absorbed and distributed throughout the body. Cyanide is primarily metabolized to thiocyanate by thiocyanate esterase or 3-mercaptopyruvate thiotransferase. Cyanide metabolites are excreted in the urine. (L96)

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Biological Half-Life
After oral administration of radiolabeled flonicamid, the radiolabeled substance is rapidly and extensively absorbed (approximately 80% of the administered dose), widely distributed, extensively metabolized, and rapidly excreted, primarily via feces (approximately 80%) and bile (approximately 70%), with a small amount excreted via urine (approximately 20%). Peak plasma concentration is reached within 1 hour after administration. The elimination process is biphasic, with a first-phase half-life of 2 to 5 hours and a second-phase half-life of 30 to 60 hours.

[1]. effect of phenylpyrrole fungicide fludioxonil on morphological and physiological characteristics of Sclerotinia sclerotiorum. Pesticide Biochemistry and Physiology Volume 106, Issues 1-2, May-June 2013, Pages 61-67.

Fludioxonil is a benzodioxane compound with the molecular formula 2,2-difluoro-1,3-benzodioxane, substituted at the 4-position with a 3-cyanopyrrole-4-yl group. It is a fungicide used for seed treatment to control a variety of diseases, including Fusarium, Rhizoctonia, and Alternaria. Fludioxonil exhibits dual action as an androgen antagonist, estrogen receptor agonist, and antifungal pesticide. It belongs to the benzodioxane, pyrrole, nitrile, and organofluorine compounds. Fludioxonil is a fungicide used for seed treatment to control a variety of diseases, including Fusarium, Rhizoctonia, and Alternaria. It has non-systemic action and long residual activity. It also inhibits glucose transport-associated phosphorylation, thereby inhibiting mycelial growth.

C12H6F2N2O2

248.1888

248.039

131341-86-1

86398

White to off-white solid powder

1.6±0.1 g/cm3

420.4±45.0 °C at 760 mmHg

199.4°

208.0±28.7 °C

0.0±1.0 mmHg at 25°C

1.622

3.67

1

5

1

18

382

0

MUJOIMFVNIBMKC-UHFFFAOYSA-N

InChI=1S/C12H6F2N2O2/c13-12(14)17-10-3-1-2-8(11(10)18-12)9-6-16-5-7(9)4-15/h1-3,5-6,16H

4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile

CGA 173506; Fludioxonil

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)