Absorption, Distribution and Excretion
Rapidly absorbed, widely distributed and mostly excreted within 48 hr.
Groups of Wistar Hsd/Cpb: Wu rats (about 200 g at treatment) were dosed with labeled spirodiclofen (radiopurity > 98%) in 10 mL/kg of 0.5% CMC suspension as follows (showing group designations in brackets): [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] 14 daily doses with 2 mg/kg/day non-radioactive a.i., then 1 labeled low dose (2 mg/kg, 4 M); and [13] single low dose, bile cannulation study (2 mg/kg, 6 M). In low dose groups, about 70% of administered dose was absorbed, with most of the label found in the urine, and about 12% of administered dose found in the bile. In the high dose (100 mg/kg) group, 61% of label was found in feces, vs. 35% in urine, suggesting saturable absorption at high dose levels. Very little label (0.05% of administered dose) was found in exhaled CO2. Peak plasma concentrations were observed between 2.5 hr to 3.9 hr in low dose groups, vs. 5.6 hr in high dose rats.
This study employed 2 male monkeys, each dosed with about 0.2 mg/kg spirodiclofen in a single treatment. The iv treatment was prepared as a PEG 200 solution in water, and the dermal treatment was a suspension of fine spirodiclofen crystals in water. The patch for the dermal treatment was removed after 8 hr, after which the application area was washed with 1% Ivory detergent solution followed by tape stripping and alcohol swab wiping. Monkeys were maintained in metabolism cages after dosing (except that the first 8 hr after the iv treatment was spent in a primate chair). Following iv dosing, urinary excretion was rapid: 64% of administered dose was obtained in urine within the first 8 hr, with an additional 18% in the next 16 hr. A total of 87% of dose was obtained in urine, and an additional 15% in cage debris/rinse (attributed primarily to urine). About 5% of administered dose was found in feces in the iv test. Measured recovery was slightly more than theoretical. Following dermal treatment, 1.1% of administered dose was found in urine, 0.3% in cage wash, and 0.2% in feces. Most of the dermally administered dose was found in the detergent swab process. An additional 9% was found in the patch or containment dome, and 10% was obtained with the alcohol swab step. Thus the dermal response from this one subject suggested only about 1.6% total absorption.
This study employed 5 male monkeys, each dosed dermally with an average of 0.04 mg/kg spirodiclofen as the SC 240 formulation in a single treatment, considered to represent a plausible field exposure level. About 2.0% of administered dose was recovered in urine plus cage rinse and other collected label attributed to urine. About 0.1% of administered dose was found in feces. Most of the material balance was found in skin wash soap swabs or ethanol extracts of the swabs (>74% of administered dose), plus small additional amounts in the patch, patch securing materials, tape strips, and alcohol swabs. Thus absorption was determined to be about 2.1% of administered dose.

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Metabolism / Metabolites
Metabolism in rats and ruminants involves cleavage of the ester, followed by hydroxylation of the cyclohexane ring. In rats, metabolism continues with cleavage of the enol ring, leading to formation of the cyclohexyl ester of 2,4-dichloromandelic acid, which is further metabolised. The residue definition comprises spirodiclofen enol.
The primary initial product of ester cleavage (removing a 2,2-dimethylbutyric acid moiety) is designated BAJ 2510 (or BAJ 2740 enol). Two major products of this enol are the 4-OH and 3-OH addition products to the cyclohexyl ring, i.e. "4-OH BAJ 2510" and "3-OH BAJ 2510."
Groups of Wistar Hsd/Cpb: Wu rats (about 200 g at treatment) were dosed with labeled spirodiclofen (radiopurity > 98%) in 10 mL/kg of 0.5% CMC suspension as follows (showing group designations in brackets): 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); 14 daily doses with 2 mg/kg/day non-radioactive a.i., then 1 labeled low dose (2 mg/kg, 4 M); and single low dose, bile cannulation study (2 mg/kg, 6 M). ... There was a sex difference in urinary metabolites: low dose females excreted 53% of administered dose as the enol, whereas low dose males excreted low amounts of the enol (< 5%), but instead favored subsequent hydroxylation of the cyclohexyl moiety of the enol at carbon 3 or 4. Positions of the ring hydroxyls in the plain of the ring (designated "e" for equitorial) were most abundant: low dose males had 26% to 30% of administered label as the 3-hydroxy-enol (e) metabolite, and 13% to 15% of administered label as the 4-hydroxy-enol (e) metabolite. The associated axial "a" isomers with the hydroxyls perpendicular to the ring were comparatively minor metabolites. The combined 3- and 4-hydroxy-enol metabolites in females constituted only 17% of administered dose. There were no other common urinary metabolites. Pre-treatment with unlabeled low doses of spirodiclofen for 2 weeks had no obvious effect on metabolism. Fecal metabolism yielded 1 to 4% of parent spirodiclofen after low dose exposure, compared to 16% in high dose males (consistent with reduced absorption). The enol constituted 4 to 7% of administered dose in feces of low-dose non-cannulated rats (16% in high dose M), with 3- and 4-hydroxy-enol (e) metabolites as modest contributors (1% to 7% of administered dose for each of these isomers). Fecal metabolites included a few percent of mandelic acid-cyclohexyl-methyl esters (created by oxidatively opening the 5-membered ring at the location of the enol hydroxyl group), and subsequent metabolic products. Glucuronides were not observed in feces. The two most common bile residues were the OH-enol glucuronide and 3-hydroxy-enol (e) (3% and 4% of administered dose, respectively).
At week 20, blood samples were taken from 4 dogs per sex at the high dose (600 ppm) at 0, 2, 4, 7, and 24 hours after feeding. Plasma concentrations of BAJ 2740 and the metabolite BAJ 2510 were evaluated by high performance liquid chromatography (HPLC). BAJ 2740 was below the limit of quantification since it was rapidly cleaved by esterases in plasma and liver to the metabolite BAJ 2510. No other metabolites were identified. Week 20 high dose group mean concentrations of metabolite BAJ 2510 in plasma were 24.8, 17.6, 19.1, 26.7, and 32.4 nmol/mL for males and 26.8, 15.9, 15.8, 25.0, and 28.1 nmol/mL in females at 0, 2, 4, 7, and 24 hours after feeding respectively. BAJ 2510 was also quantified in urine samples taken from 3 female and 1 male high dose (600 ppm) dogs at week 28. One hour after receiving treated diet, dogs were placed in metabolism cages for 5 hours. Urine volumes were 74, 281, and 305 mL in females and 18.6 mL in the male. BAJ 2510 concentrations in urine were 0.46, 0.16, and 0.12 umol/mL in females and 0.05 umol/mL in the male respectively.

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Biological Half-Life
The half life of spirodiclofen was investigated using spiked rat plasma; it was estimated to be about 15 minutes.
Groups of Wistar Hsd/Cpb: Wu rats (about 200 g at treatment) were dosed with labeled spirodiclofen (radiopurity > 98%) in 10 mL/kg of 0.5% CMC suspension as follows (showing group designations in brackets): 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); 14 daily doses with 2 mg/kg/day non-radioactive a.i., then 1 labeled low dose (2 mg/kg, 4 M); and single low dose, bile cannulation study (2 mg/kg, 6 M). ... Plasma radioactivity typically dropped about 10-fold in all groups between 8 hr and 24 hr after dosing (plasma phase 1 elimination t 1/2 values were 2.4 hr to 4.2 hr). This is consistent with swift clearance from organs and tissues as previously reported. ...

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

Spirodiclofen can cause cancer according to The Environmental Protection Agency (EPA).
Spirodiclofen is an organochlorine acaricide, a dichlorobenzene, an oxaspiro compound and a gamma-lactone. It is functionally related to a 1,3-dichlorobenzene.

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Mechanism of Action
Testicular mitochondrial preparations were evaluated for side chain cleavage of 25-OH cholesterol to pregnenolone (by assaying for progesterone after a subsequent oxidation step). In a mitochondrial preparation supplemented with NADP and in an environment of low malate levels (0.5 mM); 100 uM and 300 uM BAJ 2510 reduced progesterone synthesis to 68% and 24% of control groups, respectively. In contrast, spirodiclofen, 4-OH BAJ 2510, and 3-OH BAJ 2510 at concentrations up to 100 uM or (in the case of spirodiclofen, at the limits of solubility) had little or no effect on progesterone synthesis. When 0.5 mM citrate (and no malate) was present as a substrate (citrate also being capable of reducing NAD), even 300 uM BAJ 2510 had no remarkable effect on progesterone synthesis. This suggested an interference of BAJ 2510 with the Krebs cycle related to malate dehydrogenase activity. This was confirmed when investigators evaluated the oxidation of NADH due to malate dehydrogenase activity (assessing activity from both mitochondrial and cytoplasmic fractions): there was a clear dose-responsive inhibition of such activity due to BAJ 2510 concentrations in the 1 to 100 uM range (mitochondrial) or the 10 to 300 uM range (cytoplasmic). In contrast, BAJ 2510 had no effect on malic enzyme activity (assessed by NADP reduction with malate as substrate). In a dynamic organ culture of testicular tissue (6 hr incubation with steroidogenesis stimulated by 1 IU/mL hCG), BAJ 2510 concentrations of 10 to 300 uM caused marked, dose-related decrements in testosterone in both the tissue pieces and in the medium. An early step in progesterone synthesis from 25-OH cholesterol was markedly inhibited by BAJ 2510 in mitochondrial preparations. In contrast, progesterone levels were not statistically significantly reduced at any level with BAJ 2510 in the dynamic organ culture system with testicular tissue. As a positive control, ketoconazole profoundly reduced testosterone in tissues and medium, also without significantly reducing the quantity of progesterone in the tissue pieces. Thus it appears that BAJ 2510 toxicity is related to interference with cellular energy metabolism. /BAJ 2510, metabolite/

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.)