Absorption, Distribution and Excretion
Absorption is rapid and distribution is widespread, with most of the drug being excreted within 48 hours. Wistar Hsd/Cpb:Wu rats (approximately 200 g at treatment) were administered the following: 10 mL/kg of labeled spirodiclofen in 0.5% CMC suspension (radioactive purity > 98%) (group numbers in parentheses): [6] Single high dose (100 mg/kg, 4 M); [7] Single low dose (including CO2 measurement) (2 mg/kg, 4 M); [8] Single low dose (EPA basic test) (2 mg/kg, 4 M); [9] Single low dose (2 mg/kg, 4 F); [10] 2 mg/kg/day of non-radioactive drug for 14 consecutive days, followed by a single labeled low dose (2 mg/kg, 4 M). [13] Single low-dose bile duct cannulation study (2 mg/kg, 6 male monkeys). In the low-dose group, approximately 70% of the administered dose was absorbed, with most of the markers present in urine and approximately 12% in bile. In the high-dose group (100 mg/kg), 61% of the markers were present in feces, compared to only 35% in urine, indicating saturation absorption at high dose levels. Only trace amounts of the markers (0.05% of the administered dose) were detected in exhaled CO2. Peak plasma concentrations in the low-dose group occurred between 2.5 and 3.9 hours, while peak plasma concentrations in the high-dose group rats occurred at 5.6 hours. This study used two male monkeys, each receiving a single dose of approximately 0.2 mg/kg of spirodiclofen. The intravenous administration was prepared as a PEG 200 aqueous solution, and the dermal administration was prepared as a spirodiclofen crystal suspension. The dermal patch was removed after 8 hours, followed by cleaning the administration site with 1% ivory detergent solution, peeling off with adhesive tape, and finally wiping with an alcohol swab. Following administration, monkeys were placed in metabolic cages (except for the first 8 hours after intravenous injection, when they were placed in primate chairs). Following intravenous administration, the drug was rapidly excreted in the urine: 64% of the administered dose was excreted in the urine within the first 8 hours, followed by another 18% within the next 16 hours. A total of 87% of the administered dose was excreted in the urine, with an additional 15% present in cage debris/rinsing solution (primarily attributable to urine). In the intravenous administration test, approximately 5% of the administered dose was detected in the feces. The measured recovery rate was slightly higher than the theoretical value. Following skin administration, 1.1% of the administered dose was detected in the urine, 0.3% in the cage cleaning solution, and 0.2% in the feces. The majority of the skin-administered dose was present during the cleaning swab process. An additional 9% was present in the patch or sealing cap, and 10% was present during the alcohol swab step. Therefore, the subject's skin response indicates an overall absorption rate of only approximately 1.6%.
This study used five male monkeys, each receiving a single transdermal administration of spirodiclofenac SC 240 formulation at a mean dose of 0.04 mg/kg, which was considered to represent a reasonable level of field exposure. Approximately 2.0% of the administered dose was recovered from urine, cage cleaning solutions, and other samples collected labeled as urine. Approximately 0.1% of the administered dose was detected in feces. The majority of the drug was present in the ethanol extract of skin cleaning soap swabs or swabs (more than 74% of the administered dose), with smaller amounts also present in patches, patch fixation materials, tape, and alcohol swabs. Therefore, the absorption was approximately 2.1% of the administered dose.
Metabolism/Metabolites
In rats and ruminants, drug metabolism involves the cleavage of the ester bond followed by hydroxylation of the cyclohexane ring. In rats, metabolism continues, with the enol ring cleaved to generate cyclohexyl 2,4-dichloromandelate, which is further metabolized. The residue definition includes spirodiclofenac. The major initial product of ester bond cleavage (with the 2,2-dimethylbutyric acid moiety removed) is designated as BAJ 2510 (or BAJ 2740 enol). The two major products of this enol are 4-OH and 3-OH addition products on the cyclohexyl ring, namely "4-OH BAJ 2510" and "3-OH BAJ 2510". Wistar Hsd/Cpb:Wu rats (approximately 200 g at treatment) were administered labeled spirodiclofen (radioactive purity > 98%) in a 0.5% CMC suspension at 10 mL/kg (group names in parentheses): single high dose (100 mg/kg, 4 M); single low dose (including CO2 measurement) (2 mg/kg, 4 M); single low dose (EPA basic test) (2 mg/kg, 4 M); single low dose (2 mg/kg, 4 F); non-radioactive ai at 2 mg/kg/day for 14 consecutive days, followed by a single labeled low dose (2 mg/kg, 4 M). Single low-dose bile duct cannulation study (2 mg/kg, 6 months). …Sex differences were observed in urinary metabolites: low-dose females excreted 53% of the administered dose in enol form, while low-dose males excreted less (<5%), but were more prone to hydroxylation of the cyclohexyl moiety of the enol form at carbon positions 3 or 4. The highest concentration of hydroxyl groups (labeled “e”, indicating equatorial position) was observed in the ring plane: 26%–30% of the drug-labeled markers in low-dose males were present as 3-hydroxyenol (e) metabolites, and 13%–15% as 4-hydroxyenol (e) metabolites. The “a” isomer, with the hydroxyl group perpendicular to the ring axis, was relatively rare. In female rats, the total 3- and 4-hydroxyenol metabolites accounted for only 17% of the administered dose. No other common urinary metabolites were detected. Pretreatment with unlabeled low-dose spirodiclofen for 2 weeks had no significant effect on metabolism. Following low-dose exposure, spirodiclofen precursors produced in feces accounted for 1% to 4%, while in high-dose male rats it was 16% (consistent with reduced absorption). In the feces of low-dose unintubated rats, enols accounted for 4% to 7% of the administered dose (16% in high-dose male rats), with smaller contributions from 3- and 4-hydroxyenol(e) metabolites (each isomer accounting for 1% to 7% of the administered dose). Fecal metabolites included several percentage points of cyclohexylmandelate (generated by ring-opening oxidation of the five-membered epoxide at the enol hydroxyl position) and its subsequent metabolites. Glucuronides were not detected in feces. The two most common residues in bile were hydroxyenol glucuronide and 3-hydroxyenol(e) (3% and 4% of the administered dose, respectively).
At week 20, blood samples were collected from four dogs of each sex in the high-dose group (600 ppm) at 0, 2, 4, 7, and 24 hours after feeding. The concentrations of BAJ 2740 and its metabolite BAJ 2510 in plasma were determined by high-performance liquid chromatography (HPLC). Because BAJ 2740 is rapidly hydrolyzed to its metabolite BAJ 2510 by esterases in plasma and liver, its concentration was below the limit of quantification. No other metabolites were detected. At week 20, the mean plasma concentrations of BAJ 2510 in male dogs in the high-dose group were 24.8, 17.6, 19.1, 26.7, and 32.4 nmol/mL at 0, 2, 4, 7, and 24 hours post-feeding, respectively, and in female dogs, they were 26.8, 15.9, 15.8, 25.0, and 28.1 nmol/mL, respectively. Furthermore, at week 28, quantification of BAJ 2510 was performed on urine samples from three female and one male high-dose (600 ppm) dogs. The dogs were placed in metabolic cages for 5 hours one hour after receiving the treated feed. Urine volumes in female rats were 74, 281, and 305 mL, respectively, while the urine volume in male rats was 18.6 mL. The concentrations of BAJ 2510 in the urine of female rats were 0.46, 0.16, and 0.12 μmol/mL, respectively, while the concentration in the urine of male rats was 0.05 μmol/mL.

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Biological Half-Life
The half-life of spirodiclofen was studied using rat plasma supplemented with spirodiclofen; it was estimated to be approximately 15 minutes.
Wistar Hsd/Cpb:Wu rats (approximately 200 g at treatment) were administered the following doses: a single high dose (100 mg/kg, 4 M); a single low dose (including CO2 measurement) (2 mg/kg, 4 M); a single low dose (EPA baseline test) (2 mg/kg, 4 mg/mL); a single low dose (2 mg/kg, 4 mg/mL); 2 mg/kg/day of non-radioactive drug 14 times daily, followed by a single labeled low-dose test (2 mg/kg, 4 mg/mL); and a single low-dose bile duct cannulation study (2 mg/kg, 6 mg/mL). …Within 8 to 24 hours post-administration, plasma radioactivity generally decreased by approximately 10-fold in all groups (plasma phase I elimination half-life of 2.4 to 4.2 hours). This is consistent with previously reported results of rapid drug clearance from organs and tissues. …

Non-Human Toxicity Values
Rat inhalation LC50 >5000 mg/cu m/4 hr; Rat dermal LD50 >2000 mg/kg; Rat oral LD50 >2500 mg/kg.

[1]. The multiple target use of spirodiclofen (Envidor 240 SC) in IPM pomefruit in Belgium. Commun Agric Appl Biol Sci. 2009;74(1):225-232.

According to the U.S. Environmental Protection Agency (EPA), spirodiclofen may be carcinogenic. Spirodiclofen is an organochlorine acaricide belonging to the dichlorobenzene, oxaspirocyclic, and γ-lactone classes. Its structure is similar to 1,3-dichlorobenzene.

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Mechanism of Action
We evaluated the cleavage of the 25-hydroxycholesterol side chain in a testicular mitochondrial formulation to generate pregnenolone (progesterone content was determined by subsequent oxidation steps). In mitochondrial formulations with added NADP and low malate levels (0.5 mM), 100 μM and 300 μM BAJ 2510 reduced progesterone synthesis to 68% and 24% of the control group, respectively. Conversely, spirodiclofen, 4-hydroxyBAJ 2510, and 3-hydroxyBAJ 2510 had little effect on progesterone synthesis at concentrations up to 100 μM (spirodiclofen even reaches its solubility limit). When 0.5 mM citrate (excluding malate) was used as a substrate (citrate can also reduce NAD), even concentrations of BAJ 2510 up to 300 μM had no significant effect on progesterone synthesis. This suggests that BAJ 2510 may interfere with the Krebs cycle associated with malate dehydrogenase activity. Researchers confirmed this by assessing NADH oxidation induced by malate dehydrogenase activity (evaluating the activity of mitochondrial and cytoplasmic components): BAJ 2510 concentrations at ranges of 1–100 μM (mitochondria) or 10–300 μM (cytoplasm) showed significant dose-dependent inhibition. Conversely, BAJ 2510 had no effect on malate dehydrogenase activity (assessed via NADP reduction using malate as a substrate). In dynamic organ cultures of testicular tissue (stimulated with 1 IU/mL hCG for 6 hours), concentrations of BAJ 2510 at concentrations of 10–300 μM resulted in a significant, dose-dependent reduction in testosterone levels in both tissue blocks and culture medium. In mitochondrial preparations, BAJ 2510 significantly inhibited the early steps of progesterone synthesis from 25-hydroxycholesterol. Conversely, in dynamic organ culture systems of testicular tissue, BAJ 2510 did not cause a statistically significant decrease in progesterone levels at any concentration. As a positive control, ketoconazole significantly reduced testosterone levels in tissues and culture media, but did not significantly reduce progesterone content in tissue blocks. Therefore, the toxicity of BAJ 2510 appears to be related to interference with cellular energy metabolism. /BAJ 2510, metabolites/

C21H24CL2O4

411.32

410.105

148477-71-8

177863

White to off-white solid powder

1.3±0.1 g/cm3

561.1±50.0 °C at 760 mmHg

94.8ºC

199.8±29.1 °C

0.0±1.5 mmHg at 25°C

1.571

6.47

0

4

5

27

634

0

DTDSAWVUFPGDMX-UHFFFAOYSA-N

InChI=1S/C21H24Cl2O4/c1-4-20(2,3)19(25)26-17-16(14-9-8-13(22)12-15(14)23)18(24)27-21(17)10-6-5-7-11-21/h8-9,12H,4-7,10-11H2,1-3H3

[3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl] 2,2-dimethylbutanoate

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)

DMSO : ~120 mg/mL (~291.74 mM)
Ethanol : ~120 mg/mL (~291.74 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 3 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 3 mg/mL (7.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 3 mg/mL (7.29 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 3 mg/mL (7.29 mM) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 6: ≥ 3 mg/mL (7.29 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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