spirodiclofen (ISO);3-(2,4-dichlorophenyl)-2-oxo-1-oxaspiro[4.5]dec-3-en-4-yl 2,2-dimethylbutyrate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

albino male and female rats derived from Rattus norvegicus.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rationale: recognised as an applicable basic test system for the evaluation of possible hazard to human health
Breeder: Harlan-Winkelmann GmbH, 33178 Borchen, Germany
Supplier: Animals of all 4 groups (study no. T 2062202) were delivered ca.10 days
prior to the radioactive administration and received the same test group number in the study M 01819074
Number: 8 males, 8 females
Age: 4 – 5 weeks at start of subchronic feeding
14-15 weeks at the administration of the radioactive compound
Weight: males: 401 - 462g; females: 215 - 286g at the radioactive administration
Acclimation: The animals were acclimated to the laboratory conditions in Makrolon cages in the
Institute for Metabolism, Monheim, for about 10 days prior to the start of the experiments. They
were fed ad libitum with their pre- treatment diet until the radioactive administration.
Identification: cage cards and coloured spots on the tail.

Route of administration:

oral: feed

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

suspended in 0.5%

Details on exposure:

For the preparation of the administration suspension an adequate portion of the stock solution was pipetted and blown dry under a gentle stream of nitrogen. The dry residue was reconstituted in 0.33 mL acetone/acetic acid (98.5/1.5; v/v) and afterwards suspended in a 0.5% aqueous Carboxymethyl cellulose (CMC) solution (10.67 mL, CMC / acetic acid (99.5 / 0.5, w/v) by ultrasonication in a water bath at ca. +70 °C for about 30 min. The suspension was stirred overnight at room temperature.
In accordance with the results of the previous study [1], the dose was fixed at 2- mg/kg bw Identical volumes of the test suspensions were administered to each animal within a test group. The volume was based on the average animal weight. The concentration of each administration suspension (2 mL per animal) was calculated to reach an administered amount of about 2-mg parent compound per kg rat. Due to different animals weights at administration (see Appendix 1), the actual dose varied slightly with the body weight.

Duration and frequency of treatment / exposure:

The rats have been subchronically fed with 50 and 2500 ppm of the non-radiolabelled spirodiclofen in their diet for about 15 weeks prior to the administration of the radiolabelled test compound.

Dose / conc.:

50 ppm

Remarks:

pre-treatment - non radiolabeled test material

Dose / conc.:

2 500 ppm

Remarks:

pre-treatment - non radiolabeled test material

Dose / conc.:

2 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

4 males and 2 females were pretreated with 50ppm of non-radiolabeled test material and 3 males and 4 females were pre-treated with 2500 ppm of non radiolabeled test material.

Details on study design:

Radiolabeled test material was administered orally in four separate tests to male and female rats at the dose level of 2-mg/kg body weight (bw). The rats have been subchronically fed with 50 and 2500 ppm of the non-radiolabelled test material intheir diet for about 15 weeks prior to the administration of the radiolabelled testcompound. Plasma was collected in dependence on time after the radioactive administration, in short intervals at the beginning and longer ones later on, and radioassayed. Urine was collected in the time intervals 0 – 4 h, 4 – 8 h, 8 – 24 h and 24 – 48 h and faeces daily, and radioassayed. Urine was analysed directly and faeces after extraction with suitable solvents, both by HPLC.

This study investigates the toxicokinetics of spirodiclofen following sub-chronic dietary administration to the rat. The study should be reported as supporting.

Conclusions:

The results of this study describe the biokinetics and metabolism of spirodiclofen in male and female rats concerning the absorption, distribution, excretion and biotransformation. The rats have been pre-treated with 50 and 2500 ppm spirodiclofen in their diet for about 15 weeks prior to the administration of the radiolabelled test substance. The results of this study were compared to the results of a previous study obtained with untreated rats. The biokinetic behaviour (absorption, distribution and excretion) in all four test
groups was comparable to the untreated male and female rats and was
characterised by low plasma levels and short half lives for the absorption and
elimination of the parent compound and metabolites. Only negligible radioactivity was
observed in the residual carcass and more than 95% of the recovered radioactivity
was found in the urine and faeces two days after administration. Therefore, an
accumulation potential of the parent compound and/or metabolites could be
excluded.
Radioassays and chromatographic analyses of the metabolites in the excretion
products (urine and faeces) did not show any new metabolites or degradation ways.
The metabolic pattern was nearly identical with the results obtained with the
untreated rats. Also in this study, a sex specific difference in the quantitative pattern
of metabolites was recognised. The predominant metabolite of the female rats was
the enol, which was excreted mainly via the urine.

Executive summary:

Spirodiclofen was administered orally in four separate tests to male and female rats at the dose level of 2 mg/kg bw. The rats had
been subchronically fed with 50 and 2500 ppm of the non-radiolabelled spirodicofen in their diet for 5 weeks prior to the administration of the  radiolabelled test compound.  Plasma was collected in dependence on time after the radioactive administration, in short intervals at the beginning and longer ones later on, and radioassayed.  Urine was collected in the time intervals 0-4 hours, 4 -8 hours, 8-24 hours and 24-48 hours; and faeces daily, and radioassayed. Urine was analysed directly and faeces after extraction with suitable solvents, both by HPLC. The metabolite pattern in the HPLC chromatograms was compared with those taken from a previous study in non pre-treated rats.  The plasma curves were comparable between all test groups.  Absorption from the gastrointestinal tract started immediately after the administration. The absorption half-lives ranged from 0.6-1.3 hours. The plasma concentrations of male and female rats pre-treated with 50 ppm reached maximum values of 2.1-3.3 μg/g.  Male and female rats pre-treated with 2500 ppm reached slightly lower maximum equivalent concentrations in plasma of 1.6 and 1.7 μg/g. The maximum equivalent
concentrations were measured during the period of about 3 - 8 hours after the
administration.  Spirodiclofen and its metabolites were well distributed into the organs and tissues.  Under steady state conditions the administered compound permeated readily into the tissues with a low mean residence time. This indicated that redistribution from body tissues to plasma prior to elimination was a fast  process.  The decrease of the plasma concentrations following the relatively broad
maximum of the plasma curves was characterised by short elimination half-life of 3.5-5.3 hours.  Recoveries of the administered radioactive test compound were 86% or higher.  The radioactivity was excreted fast and nearly completely. More than 97.8% of the recovered radioactivity was excreted within 48 hours after administration. The major amount of radioactivity was excreted via the urine (65.4-77.0%) and in the faeces 22.3-32.4%. The radioactivity remaining in the body excluding the gi-tract at sacrifice (48 hours after the administration) were  low (0.2-1.0%).  The duration and dose of the subchronic feeding did not  significantly influence the biokinetics and excretion of spirodiclofen in male and female rats.  According to chromatographic analysis, the parent compound was not detected in the urine. It accounted for 0.4-3.4% of the recovered radioactivity in the faeces of rats pre-treated with 50 ppm, and 5.7-15.8% in the faeces of rats pre-treated with 2500 ppm.  A series of metabolites was formed, 11 of which were identified. The identification rate was similar in all tests, ranging from 91.4-94.1% of the recovered dose (80.8-95.6% of the administered dose).  The main metabolites (> 10%) were the enol and two diastereoisomers each of
3- and 4-hydroxy-enol with the hydroxy group in the equatorial (e) and the axial (a) position. Sex-specific differences in the amounts of the excreted main metabolites were observed. The predominant metabolites in the excreta of male rats were the equatorial isomers of 3-and 4-hydroxy-enol (e) accounting for 21-35% of the recovered dose. Further metabolites were the enol accounting for 8-11% and the axial isomers of 3- and 4-hydroxy-enol (a) accounting for 6-8%. In the excreta of female rats, the main metabolite was the enol accounting for 51-55%. The 4-hydroxy-enol (e) (13-18%), the 3-hydroxy-enol (e) (5–6%), and the 3- and 4-hydroxy-enol (a) accounted for 2-4%.  For all other metabolites low quantities (< 5%) were measured.  Sub-chronic feeding of rats with 50 or 2500 ppm spirodiclofen for about 15 weeks did not significantly influence the  metabolism of spirodiclofen. The excreta of untreated and sub-chronically fed male and female rats, respectively, had a comparable metabolite pattern.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

tissue distribution

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Remarks:

14C-labelled test compound.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species: albino male rats derived from Rattus norvegicus

Sex:

male

Details on test animals or test system and environmental conditions:

Strain: Wistar Hsd/Cpb: WU
Rationale: recognised as an applicable basic test system for the evaluation of possible hazard to human health
Breeder: Harlan-Winkelmann GmbH, D-33178 Borchen, Germany
Number: 6 males
Age: 7 to 8 weeks at time of deliverance
Weight: males: about 200 g at the radioactive administration
Acclimation: The animals were acclimated to the laboratory conditions in Makrolon cages in the Institute for Metabolism, Monheim, for about 7 days prior to the administration.
Identification: cage cards and coloured spots on the tail

Route of administration:

oral: unspecified

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

suspended in a 0.5%

Details on exposure:

single oral administration

Duration and frequency of treatment / exposure:

48 hours following a single oral administration.

Dose / conc.:

3 mg/kg bw/day

No. of animals per sex per dose / concentration:

6 males

Control animals:

yes

Positive control reference chemical:

3 mg of the non-radiolabelled test compound was suspended in 10 mL of 0.5% aqueous CMC in the same manner as done for the radiolabelled compound. From this 2 mL was administered.

Details on dosing and sampling:

For the preparation of the administration suspension an adequate portion of the stock solution was transferred with a pipette and blown dry under a gentle stream of nitrogen. The dry residue was reconstituted in 0.33 ml acetone/acetic acid (98.5/1.5; v/v) and afterwards suspended in a 0.5% aqueous Carboxymethyl cellulose (CMC) solution (10.67 mL, CMC / acetic acid (99.5 / 0.5, w/v) by ultrasonication in a water bath at ca. +70 °C for about 30 min. The suspension was stirred overnight at room temperature.
The administered dose was fixed at 3-mg/kg and the volume 10-mL/kg body weight (bw). Based on an average animal weight of 200 g, the concentration of the test compound administered to each animal was 0.6-mg/2 ml_. From the stock solution, 4 mg was suspended in 13.3 ml of the CMC-solution.

Type:

excretion

Results:

highly effective excretion of radioactivity via urine and faeces

Type:

absorption

Results:

readily absorption of the radioactivity from the gastrointestinal tract

Type:

distribution

Results:

smoothly distribution of the radioactivity within the body with preference to the
liver as the main metabolising organ and the kidney as the main organ
responsible for the excretion.

Details on distribution in tissues:

As a result, concentrations in all tissues, organs or subparts thereof were obtained at different times after oral dosage of 3 mg/kg bw spirodiclofen to describe the kinetic behaviour of the administered radioactivity in quantitative terms. These distribution patterns can be attributed to the unchanged parent compound and metabolites.
A steady decline in the equivalent concentration was observed for all organs and tissues from 1 to 48 hours after dosage (Figures 8-11 and Table 1). Generally, the concentrations (up to 6 ug/g) were highest in the small intestine, liver, kidney, and urinary bladder. Beside these important organs which are responsible for the degradation and excretion of spirodiclofen and metabolites, significant higher
radioactivity was detected in the brown fat tissue from 1 to 8 hours after administration (2.7 ug/g after 4 hours). Important was the result that the glandular organs like adrenals, thyroid, or testes had no higher values than 1 ug/g. However, in all cases the values dropped by several orders of magnitude during the test period of 48 hours. There was no sign of accumulation in any one of the organs or tissues.

Metabolites identified:

not measured

REACH information requirements are limited to a theoretical assessment; however, data are available and should be reported.

Conclusions:

The kinetic behaviour (measured over 48 hours) of the new acaricide spirodiclofen in the rat after a single oral administration of the [dihydrofuranone-3-14C] radiolabelled compound (3 mg/kg bw) can be characterised by the following facts:
• readily absorption of the radioactivity from the gastrointestinal tract,
• smoothly distribution of the radioactivity within the body with preference to the liver as the main metabolising organ and the kidney as the main organ responsible for the excretion,
• extrabiliary secretion of radioactivity into specific organs or tissues (e.g. into the small intestine, stomach and deeper skin layers),
• no intensive blackening of glandular organs or tissues and those which are involved in the regulation of hormones (e.g. adrenal, testis, or thyroid gland),
• no accumulation of radioactivity in specific organs or tissues during test period,
• highly effective excretion of radioactivity via urine and faeces and
• nearly completely bioavailability of the parent compound and metabolites to the animal.
The results of this study clearly demonstrate that the quantitative whole body autoradiography (QWBA) can provide detailed data on the kinetics of the absorption and distribution of the total radioactivity in the organs and tissues of the rat. Therefore, this new technology can be used together with all other ADME studies (absorption, distribution, metabolism, and excretion) to find a detailed picture on the administered xenobiotic in the rat.

Executive summary:

The distribution of the spirodidlofen, ¹⁴C-labelled in the 3-position of the dihydrofuranone ring of the molecule, was investigated by quantitative radioluminography (RLG).  The data were obtained over a period of 48 hours following single oral administration of a dose level of 3 mg/kg bw.  Spirodiclofen was readily absorbed from the gastrointestinal tract, after degradation to the initial metabolite (enol).  One hour after administration, radioactivity was found in all organs and tissues.  The high levels radioactivity in the liver and kidney indicated that further degradation to other metabolites and renal excretion, respectively, had already begun. This observation was in good agreement with the higher polarity and water solubility of the metabolites. The uniform blackening of the small intestine one hour after administration and the significant higher radioactivity of the mucous membrane of the stomach and the deeper skin layers seen after eight hours originated from an extrabiliary secretion of radioactivity into these tissues.  At later stages in the study (24 and 48 hours post administration(, the most prominent feature were still the excretory organs (liver, kidney and large intestine).  There was no sign of accumulation of radioactivity in specific organs or tissues (e.g. brown fat tissue). Radioactivity was not detected in organs which are responsible for the hormonal regulation (i.e. adrenal gland, testis, thyroid gland) after a single oral administration.  Nearly complete bioavailability can be assumed. For all organs and tissues, a steady decline in the equivalent concentrations was observed from 1 to 48 hours after dosing; values dropped from 6 ug/g one hour after administration to <0.03 ug/g at the end of the test period.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: Albino male and female rats derived from Rattus norvegicus
Strain: Wistar Hsd/Cpb: Wu
Rationale: Recognised as an applicable basic test system for the evaluation of possible hazard to human health
Breeder: Harlan-Winkelmann GmbH, 33178 Borchen, Germany
Number: 22 males, 4 females
Age: 7 - 12 weeks
Weight: Approximately 200 g
Acclimation: The animals were acclimated to the laboratory conditions in plastic cages on wood shavings for at least one week prior to the start of each experiment.
Identification: Cage cards and coloured spots on the tail
Rooms: The animals were kept under conventional hygienic conditions in air-conditioned rooms at approximately 18 –25 °C and a humidity 27-65% at a 12 hours photocycle and 10-15 fold air change per hour. The animals were kept at room temperature during the test period.
Cages: During the excretion studies the animals were kept in special metabolism cages, which allowed for a separate and quantitative sampling of the excreta. During the non-radioactive pre treatment period (test no. 10), the rats were housed as single animals in plastic cages.
Diet: Altromin 1324 standard food, Fa. Altrogge, 32791 Lage/Lippe, Germany; ca. 18 g per day and animal
Water: Tap water was given ad libitum (water specification according to local drinking water regulations).
Randomisation: Randomisation was carried out by lot

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The radiolabelled compound was shipped in solid form. Upon arrival it was dissolved in acetonitrile and stored as such in a refrigerator (about +4 °C) or in a deep freezer (about -20 °C). Three different batches of radiolabelled spirodiclofen were used in tests 6 - 10 and 13.
For the preparation of the administration suspension an adequate portion of the stock solution was pipetted and blown dry under a gentle stream of nitrogen. The dry residue was solved in 0.33 mL acetone/acetic acid (98.5/1.5; v/v) and afterwards suspended in a 0.5% aqueous Carboxymethyl cellulose (CMC) solution (10.67 mL, CMC / acetic acid (99.5/0.5; w/v) by ultrasonication in a water bath at 70 °C for 30 min. The suspension was stirred overnight at room temperature. Adequate volumes of the test suspensions were administered to each rat. The concentration of each administration suspension was calculated to reach an administered amount of about 100 mg parent compound per kg body weight (bw) in test 6, 2 mg/kg bw in tests 7 – 10, and 1 mg/kg bw in test 13. As the animal weights slightly varied, the actual dose varied slightly with the body weight.

Duration and frequency of treatment / exposure:

All groups were administrated a single dose of test material, and one group had multiple low concentration doses over 14 days. The following tests are shown below.

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

single high dose experiment

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

single low dose experiment;
CO2-test

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

single low dose experiment;
EPA-basic test

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

single low dose experiment

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

multiple dosing test,
low dose

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Bile cannulation experiment;
single low dose test

No. of animals per sex per dose / concentration:

#1 to #5 - 4male rats per dose and #6 - 6 male rats

Control animals:

not specified

Positive control reference chemical:

The non-radiolabelled parent compound - spirodiclofen.

Details on dosing and sampling:

- Tissues and body fluids sampled: urine, faeces, expired 14C-carbon dioxide , blood, plasma, bile, urine and faeces in the bile-cannulation experiment, sacrifice, preparation of organs and tissues, volatility.

- Measurement of radioactivity: Measurement of Solid Samples Using Liquid Scintillators, Measurement of Liquid Samples Using Liquid Scintillators (LSC).
-Limit of Detection, Limit of Quantitation:
In addition to the experimental values, background values were determined for all measuring procedures in a separate experiment. For this purpose, “blank” samples were prepared for measurement in the same way as the experimental samples, e.g. plasma, or tissue/organ prior to the administration of the test compound. For organ/tissues samples that were solubilized in a tissue solubiliser and for all other samples, the limit of detection (LOD) was established at 10 dpm measured per aliquot after correction for the background radioactivity (see below). The limit of quantitation (LOQ) for each individual measurement was established as 2 to 3 times of the background radioactivity (dpm) of each instrument/method. The respective value was printed out on the original raw data sheet. This background counting rate was in a range between 10 – 45 cpm (approximately equal to 10 – 45 dpm) and it was automatically subtracted from the measuring results. Samples with individually measured values below two to three times the background radioactivity and for which the average counting efficiency was lower than 50% and the percentage error greater than 10% were not quantified and labelled as < LOQ in the respective tables. The only exception was the measurement of radioactivity in the expired air samples for which all reasonable dpm-values were quantified. The samples were measured generally between seconds and 20 min. depending on the amount of radioactivity present in the sample. The measurement was stopped after reaching a 2-s error of 0.7%. If this error was not reached within 20 min., the measurement was stopped and the 2-s error of the dpm-value reached at that time was printed out.

-Quantitative Evaluation: Statistical Evaluation, Data recording and Calculations, Dose-normalised Concentration P and Equivalent Concentration C, Radioactive Residues, Exponential Analysis.

- Analytical methods: Preparation of Rat Urine for Analysis and Identification of Metabolites, Preparation of Rat Faeces for Analysis and Identification of Metabolites, Preparation of Rat Bile for Analysis and Identification of Metabolites, High Performance Liquid Chromatography (HPLC), Mass Spectroscopy, NMR-Spectroscopy.

Preliminary studies:

A survey of all animal experiments is given in Table 1. The amounts of the test substance and the radioactivity administered to the rats in the different experiments are summarised in chapter 3.3.1.
In test number 6 (single oral high dose) four male rats received a single oral dose of about 100 mg spirodiclofen/kg bw. A 2-mL-portion of the administration suspension containing ca. 20 mg spirodiclofen was administered to each rat with a mean body weight of 211 g.
In test number 7 (expiration test, single oral low dose) four male rats received a single oral dose of about 2 mg spirodiclofen /kg bw. A 2-mL-portion of the administration suspension containing ca. 0.4 mg spirodiclofen was administered to each rat with a mean body weight of 210 g.
In test number 8 (single oral low dose) four male rats received a single oral dose of about 2 mg spirodiclofen /kg bw. A 2-mL-portion of the administration suspension containing ca. 0.4 mg spirodiclofen was administered to each rat with a mean body weight of 207g.

In test number 9 (single oral low dose) four female rats received a single oral dose of about 2 mg spirodiclofen /kg bw. A 2-mL-portion of the administration suspension containing ca. 0.4 mg spirodiclofen was administered to each rat with a mean body weight of 212 g.
In test number 10 (single oral multiple dose) four male rats were pre-treated with 14 daily oral doses of 2 mg unlabelled spirodiclofen /kg bw prior to the administration of a single oral dose of about 2 mg labelled spirodiclofen /kg bw. A 2-mL-portion of the administration suspension containing ca. 0.4 mg spirodiclofen was administered to each rat with a mean body weight of 219 g.
In test number 13 (bile cannulation, single oral low dose) six male rats were surgically implanted with biliary and duodenal cannulae under general anaesthesia (see below). One day after operation, the rats were applied with a single oral dose of about 1 mg spirodiclofen /kg bw. A 1-mL-portion of the administration suspensioncontaining ca. 0.2 mg spirodiclofen was administered to each rat with a mean bodyweight of 197 g.

Type:

clearance

Results:

A total clearance of 0.91 to 2.16 mL/min x kg bw was calculated for male and
female rats of all dose groups. The renal clearance ranged from 0.59 to 1.45 mL/min
x kg bw for rats from all test groups.

Type:

excretion

Results:

The overall excretion of the radioactivity was a fast and almost complete
process.

Type:

distribution

Results:

The mean residence time (MRT) of the total radioactivity in the rat body was
small in all test groups. Values of 6 - 12 hours indicated that the redistribution of the
total radioactivity into the plasma prior to the elimination was a fast process.

Type:

absorption

Results:

The lag time between administration and the onset of absorption amounted to 0.22 - 0.27 hours as calculated by the pharmacokinetic curve-fitting program. The amount of renal excretion (64 – 76 % of the recovered radioactivity)

Details on absorption:

Spirodiclofen was rapidly absorbed from the gastrointestinal tract of male andfemale rats in all test groups. The plasma curves (Figures 7 - 10) show that the absorption of the radioactivity from the GIT started immediately after the administration of the radiolabelled dose. The lag time between administration and the onset of absorption amounted to 0.22 - 0.27 hours as calculated by the pharmacokinetic curve-fitting program TOPFIT (Table 4).
The amount of renal excretion (64 – 76 % of the recovered radioactivity) in the low dose experiments indicated that the absorption rate was at least 60 % of the given dose. Based on the data of the bile-cannulation experiment in which ca. 12 % of the dose was detected in the bile, the before mentioned 60 % absorption rate value can be enhanced to at least 72 %.

Details on distribution in tissues:

After the rapid absorption of the administered radioactivity from the GIT into the plasma, a relatively broad maximum (ca. 3 h) of the plasma curves was observed in all tests (Table 3). Male rats treated with 2 mg spirodiclofen/kg bw reached plasma curve plateau concentrations of 2.32 - 2.66 µg/g 2 to 6 hours post dosing. Female rats reached equivalent concentrations of 2.01 – 2.06 µg/g 1.5 to 4 hours after the administration of 2 mg spirodiclofen /kg bw. The maximum concentrations in the plasma of male and female rats were measured 3 hours post administration and amounted to 2.66 and 2.11 µg/g, respectively. Male rats, which had received 14 daily oral administrations of 2 mg nonradiolabelled spirodiclofen /kg bw, reached equivalent concentrations in plasma of 1.70 - 2.12 µg/g 2 to 6 hours after the last radiolabelled dose. The maximum equivalent concentration of 2.12 µg/g was measured 4 hours after the last administration.
The administration of 100 mg spirodiclofen /kg bw to male rats resulted in plasma plateau concentrations of up to 43.09 – 51.34 µg/g 3 to 8 hours post administration. The maximum equivalent concentration in plasma of 51.34 µg/g was measured 8 hours post dosing. The maximum was delayed compared to the low dose experiments by ca. 4 hours giving evidence for a time dependent absorption process of the radioactivity from the GIT into the plasma. The maximum dose related concentrations P in the plasma of male and female rats following a single or repeated administration of 2 mg spirodiclofen /kg bw were 1.5 – 1.8 fold higher than the theoretical equidistribution concentration (P = 1) in the whole rat body. Since these values were higher than one, it can be assumed that the distribution of the radioactivity from the plasma into the peripheral compartments was slightly lowered. On the other hand, relative concentrations of up to 0.5, i.e. half the equidistribution concentration in case of the high dose experiment indicated asmoothly distribution of radioactivity from the plasma into the peripheral
compartments (Table 2). In all test groups, the plasma curves showed a rapid and continuous decrease of the plasma radioactivity concentrations following the maximum concentrations.
The menu driven pharmacokinetic fitting program TOPFIT was used to calculate the pharmacokinetic parameters from plasma curve analysis. The results are summarised in Table 4. The radioactivity concentrations in the plasma were subjected to two and/or three compartment
disposition modelling. Correlation coefficients varied between 0.967 and 0.999. Short absorption half lives in the range of 0.32 – 0.92 hours were calculated for the low dose experiments with male and female rats and the high dose experiment with male rats. A much more lower values (0.05 h) was found for the pre-treated rats.
These data were in the same order of magnitude as found for lag time between administration and onset of absorption (see above).
The elimination of the total radioactivity from the plasma is characterised by short half-lives of 4.25 and 3.35 for male and female rats, respectively, applying the 2-compartment model.
Applying the 3-compartment model for the curve fitting computation of the low dose female experiment, an additional second half-life was calculated for the elimination. It amounted to 6.84 hours. The comparable second half-life for the high dosed (100 mg/kg bw) male rats amounted to 132 hours. It appeared to be very long when compared to the experimentally found data. Already 168 hours after the
administration no radioactivity was measured in plasma or in the residual carcass.
The theoretical distribution volume under steady state conditions (Vss) ranged between 228 and 433 mL/kg bw for rats from all tests except the high dose experiment. Considering a total blood volume of about 64 mL/kg bw and a total body water content of about 684 mL/kg bw [15] the determined Vss values reflect a slightly lowered distribution of spirodiclofen into the peripheral organs and tissues. This effect is
already discussed for the P- values (see above). The mean residence time (MRT) of the total radioactivity in the rat body was small in all test groups. Values of 6 - 12 hours indicated that the redistribution of the
total radioactivity into the plasma prior to the elimination was a fast process.
A total clearance of 0.91 to 2.16 mL/min x kg bw was calculated for male and female rats of all dose groups. The renal clearance ranged from 0.59 to 1.45 mL/min x kg bw for rats from all test groups which was lower than the glomerular filtration rate (2.1 mL/min x kg bw).

Test no.:

#1

Transfer type:

secretion via gastric mucosa

Remarks:

the highest concentrations but on a rather low level were detected in the liver and kidney those organs, which are responsible for the metabolism and excretion.

Observation:

slight transfer

Details on excretion:

The overall excretion of the radioactivity was a fast and almost complete process. After the administration of 2 mg spirodiclofen/kg bw more than 88.3% of the administered radioactivity, i.e. more than 99.3% of the recovered radioactivity was excreted within 48 hours. About 64.4 - 76.1% of the recovered radioactivity was excreted via urine and 23.9 – 34.9% via faeces. The renal to faecal excretion ratio was 1.8 - 2.0 in single dosed male rats, 2.4 in multiple dosed male rats and 3.2 in single dosed female rats.
After the oral administration of 100 mg spirodiclofen /kg bw 96.3% of the dose or 100% of the recovered radioactivity was excreted within 168 hours. Via urine, 35.1% of the administered or 36.7% of the recovered radioactivity was excreted. Via faeces, the excretion amounted to 61.2% of the administered or 63.3% of the recovered dose. The comparably high amount of radioactivity in faeces is probably due to an uncompleted absorption of spirodiclofen from the GIT into plasma.
The elimination of radioactivity via bile was studied over a 24 hours test period using bile-cannulated rats. About 11.29% of the recovered radioactivity were detected in bile, 28.7% in faeces and 22.8% in urine.
The amounts of radioactivity excreted via urine and faeces by the individual rats from the different test groups were compared statistically using the non-parametric Whitney-Mann (Wilcoxon) test. Male rats once treated with 2 mg spirodiclofen /kg bw excreted significantly more radioactivity via faeces than female rats after a single and male rats after multiple treatment with 2 mg spirodiclofen /kg bw (probability 99%).
Consequentially, male rats once treated with 2 mg spirodiclofen /kg bw excreted significantly less radioactivity via urine than female rats after a single and male rats after multiple treatment with 2 mg spirodiclofen /kg bw.

Metabolites identified:

yes

Details on metabolites:

In this study, unknown metabolite fractions were isolated from urine, faeces, and bile by solid phase extraction (SPE) and semipreparative HPLC. From the isolated metabolite fractions 11 metabolites and the parent compound were identified by LC-MS, LC-MS/MS, and NMR investigations. Two further metabolite fractions were partially identified.

REACH information requirements are limited to a theoretical assessment; however, data are available and should be reported. This study investigates the toxicokinetics of spirodiclofen following single (low and high dose) and repeated (low dose) oral administration in rats.

 

The quantitative distribution of the identified metabolites as sum of urine, faeces
and bile is given in the following table:

Metabolites
Glyoxylic acid	0.72	1.03	1.79	0.75	1.41	0.13
Enol-OH-glucuronide	n.d.	n.d.	n.d.	n.d.	n.d.	3.40
Dichloro-mandelic acid	n.d.	0.18	0.22	0.18	0.16	0.18
metabolite U3 *)	n.d.	0.72	1.48	1.05	0.76	n.d.
3-keto-enol	0.26	0.70	0.49	n.d.	n.d.	n.d.
4-hydroxy-enol (e)	9.27	18.23	17.49	16.03	21.18	21.75
3-hydroxy-enol (a)	2.10	4.49	4.10	1.89	5.52	1.69
3-hydroxy-enol (e)	14.28	31.53	28.44	5.39	33.33	9.57
4-hydroxy-enol (a)	3.38	6.61	7.52	3.16	6.33	4.00
2-hydroxy-enol (e)	0.25	0.90	1.02	0.86	1.16	0.69
DCB-acid	1.57	0.46	1.16	0.48	0.64	0.10
2- or 3-ene-enol	0.25	0.31	0.43	0.22	0.36	n.d.
Enol	21.93	9.14	7.84	58.20	8.68	15.87
MA-cyclohexyl ester	7.55	2.45	2.94	1.33	1.64	2.14
spirodiclofen	16.04	1.83	4.26	0.66	2.32	0.67
Total identified	77.59	78.60	79.19	90.22	83.48	59.72
Total unknown	13.59	6.35	8.38	5.05	5.73	5.38
Total analysed	91.18	84.95	87.57	95.27	89.21	65.09
Loss by extraction	2.08	0.80	0.79	0.61	0.80	2.39
Total not analysed	3.03	7.28	0.00	1.60	4.22	0.00
Total radioactivity excreted	96.29	93.03	88.36	97.48	94.23	67.48

# = 14 x daily oral administration (2 mg/kg bw) of non-radiolabelled spirodiclofen prior to one final administration with the radiolabelled spirodiclofen;

n.d. = not detected; total unknown = unidentified metabolites and unassigned radioactivity; ble precursor of DCB-acid

Executive summary:

Spirodiclofen was administered orally to male and female rats at a dose level of 2 mg/kg bw.  A high dose (100 mg kg bw) and a repeated low dose experiment (2 mg/kg bw) were conducted with male rats.  In a bile-duct cannulation experiment an oral dose of 1 mg/kg bw was administered to male rats.  The objective was to study absorption, distribution, metabolism, and excretion of spirodiclofen in dependence on dose, pre-treatment, and sex. All tests were performed according to OECD 417.  Plasma curves were comparable between all test groups. The absorption of administered radioactivity from the  gastrointestinal tract started immediately after administration. Absorption half-lives ranged from 0.05-0.92 hours. The plasma curves are characterised by a relatively broad maximum lasting from about 1.5-6 hours after administration of 2 mg/kg bw. Maximum plateau concentrations of 2.0-2.6 μg spirodiclofen equivalents/g and relative concentrations (P) of 1.6-1.8 were measured. A much higher maximum equivalent concentration of 51.3 μg/g and a much lower maximum P-value of 0.6 were measured 8 hours after the administration of 100 mg/kg bw to male rats.  Spirodiclofen and its metabolites were well distributed into the organs and tissues.  Under steady state conditions the administered radioactivity permeated readily into the tissues with a low mean residence time, indicating that redistribution from body tissues to plasma prior to elimination is a rapid process. The decrease of the plasma concentrations following the broad maximum of the plasma curves is characterised by short elimination half lives. The administered radioactivity was recovered at a high rate. Recovery in the different test groups was more than 89% of the dose.  Biotransformation to volatile  metabolites was negligible (0.05% of the administered dose) showing that the selected labelling position in the molecule was metabolically stable under the in vivo conditions. The radioactivity was excreted fast and nearly completely. More than 99% of the recovered radioactivity was excreted within 48 hours after the administration of 2 mg/kg bw.  Excretion occurred via urine (66.3–76.1%) and faeces (23.9–34.9% reocvered radioactivity).  After administration of 100 mg/kg bw, 36.7% of the recovered radioactivity was excreted via urine and 63.3 via faeces.  The residues remaining in the body at sacrifice 48 hours after administration were below 1% of the administered radioactivity in rats from all low dose test groups. A similar situation was found in case of the high dose experiment with male rats, which were sacrificed 168 hours after administration. In general, the highest concentrations were detected in the liver and kidney. No significant radioactivity was found in the organs and tissues of the endocrine system, like the adrenals, thyroid, ovary and testis.  The main metabolites in all dose groups were the enol, and the equatorial and axial isomers of 3- and 4-hydroxy enol.  Sex-specific differences in the amounts of the main metabolites were observed. The most important result was the high amount (58%) of the enol compound found mainly in the urine of the females.  From bile, a glucuronide of a hydroxylated enol metabolite was identified besides the 3-hydroxy-enol (e) and traces of the other main metabolites. Due to the instability of the glucuronide, it could not be detected in faeces.  The parent compound was of minor importance in the excreta of the low dosed rats. In the excreta of rats dosed with 100 mg kg bw, the parent compound accounted for ~16% and the enol up to 22% of the administered radioactivity. These results combined with the high amount of radioactivity excreted with faeces led to the conclusion that the high amount of spirodiclofen administered to the rats was not completely absorbed.  The overall identification rate is above 70% of the administered radioactivity on average with the exception of the bile cannulation experiment. Hence, it is concluded that the metabolism of spirodiclofen in the rat is well characterised.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: To establish the levels of Spirodiclofen and the Spirodiclofen enol in the plasma and urine of dogs.
- Short description of test conditions: Spirodiclofen was administered through feed for a period of upto 28 weeks.
- Parameters analysed / observed: Concentrations of test substance and metabolites in the blood were analysed after 20 weeks and concentrations of the test substance and metabolites in urine were analysed at week 28.

GLP compliance:

no

Radiolabelling:

not specified

Species:

dog

Strain:

Beagle

Sex:

male/female

Route of administration:

oral: feed

Details on exposure:

Test substance was administered within the diet, which was available ad libitum. The frequency of treatment is not applicable as food was replenished as the test animals consumed it. Due to this, the frequency of treatment can be considered daily as the test subjects were exposed to the test substance at every meal.

Dose / conc.:

0 ppm

Remarks:

control group - plain feed only

Dose / conc.:

20 ppm

Dose / conc.:

50 ppm

Dose / conc.:

150 ppm

Dose / conc.:

500 ppm

Dose / conc.:

600 ppm

Remarks:

high dose group

No. of animals per sex per dose / concentration:

Not specified - different numbers of animals at different dose levels.

Control animals:

yes, plain diet

Details on study design:

In this part of the chronic dog study with administration of 0, 20, 50, 150 and 500/600 Spirodiclofen in the feed, blood samples were taken at week 20 from 4 animals per sex of the high dose group, which was at this time (600 ppm). Blood samples were taken at 0, 2, 4, 7 and 24 h. Plasma was obtained by centrifuging and stored at –20 °C until processing. Urine samples were taken at week 28 from 3 female and 1 male of the high dose group (600 ppm) and from one female control animal (0 ppm). The purpose of this part of the study was to determine the concentration of Spirodiclofen or its primary metabolite, Spirodiclofen enol, in plasma and urine.

Findings:

Plasma: Concentrations of Spirodiclofen in plasma were below the limit of quantification (LOQ) in all plasma samples due to the easy cleavage of Spirodiclofen by esterases in plasma and liver to the Spirodiclofen enol. With respect to the concentration of Spirodiclofen enol in plasma, there was no remarkable difference in concentrations and time course between male and female animals. The concentration of Spirodiclofen enol in plasma before feeding was with 248 nmol/mL (m) and 268 nmol/mL (f) at the same level as 7h and 24h after feeding. This finding should lead to an accumulation of the metabolite in plasma, which at week 20 could be in a steady state condition. Therefore, the concentration in plasma at week 20 should be higher than at the beginning of the study.

 

Urine: The volume of the urine samples ranged from 305 to only 19 mL. Therefore, an absolute quantification was not possible. The concentration of Spirodiclofen-enol ranged from 0.12-0.46 mmol/mL and was with 0.05 mmol/mL in the small sample of the male dog slightly lower.

Conclusions:

The easy cleavage of Spirodiclofen by esterases was previously investigated in rat plasma and liver
tissue in vitro and, therefore, it was expected that no parent compound could be measured in
plasma. The lower concentration of Spirodiclofen enol after feeding in the time period up to 4h was seen
in both sexes of dogs. This profile of concentration versus time was also seen with another
compound of the same chemical class and the same mode of action.

Executive summary:

As part of the chronic dog study with administration of 0, 20, 50, 150 and 500/600 ppm spirodiclofen, blood samples were taken at Week 20 from 4 animals per sex of the high dose group (600 ppm).  Blood samples were taken at 0, 2, 4, 7 and 24 hours afetr dosing.  Urine samples were taken at Week 28 from 3 female and 1 male dogs of the high dose group and from one female control. Plasma was obtained by centrifuging and stored at -20 °C until processing.  Due to the rapid cleavage of spirodiclofen by esterases in plasma and liver to the enol metabolite, the concentration of parent compound in all plasma samples was below the limit of quantification.  Spirodiclofen-enol was identified as the major metabolite of spirodiclofen.  There was no indication for the concentration of other metabolites.  The results of the plasma concentration reveal, that there is no remarkable difference in concentrations and time course between males and females.  The concentration of Spirodiclofen-enol in plasma before feeding was 248 nmol/mL (M) and 268 nmol/mL (F); the same level as 7 and 24 hours after feeding. This finding should lead to an accumulation of the metabolite in plasma which could at this time (Week 20) be in a steady state condition.  Therefore, the concentration in plasma at week 20 should be higher than at the beginning of the study.  In week 28 urine samples, the concentration of the metabolite Spirodiclofen-enol ranged from 0.12-0.46 umol/mL and was slightly lower in the male dog.  It was not possible to detect other metabolites by this method (HPLC/DAD).

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

This study was carried out in accordance with JMAFF Guidelines in place at the time of study initiation.

Justification for type of information:

This experimental study report is reliable for use in the risk assessment as it has been conducted by a trusted laboratory, under GLP and following JMAFF Guidelines relevant at the time of the study being conducted.

Objective of study:

toxicokinetics

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF New Test Guidelines for Supporting Registration of Chemical Pesticides 12 Nousan 8147.

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

radiolabelled in the dihydrofuranone ring of the molecule.

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

not specified

Details on exposure:

Rats given a single oral dose via gavage.

Duration and frequency of treatment / exposure:

Frequency not applicable in a single dose study.

Dose / conc.:

2 mg/kg bw (total dose)

Remarks:

male and female

No. of animals per sex per dose / concentration:

Four animals per sex per dose.

Control animals:

not specified

Details on study design:

Four animals/sex were killed at 3, 6 and 24 h. Urine and faeces were collected from time of administration to termination. Blood and tissue (liver, kidney, skin, GIT including faeces, carcass) samples were obtained at sacrifice and radioactivity levels were measured by LSC. Metabolites in urine and plasma samples were analyzed by HPLC, followed by LC-MS and LC-MS/MS. Metabolites in kidney and liver samples were analysed by HPLC.

REACH information requirements are limited to a theoretical assessment; however, data is available. This study reports the depletion of residues and metabolites in the rat after a single low dose of spirodiclofen.

Executive summary:

Spirodiclofen was administered orally to male and female Wistar rats at an oral dose of 2 mg/kg bw.  The depletion of residues from plasma, liver and kidney and the metabolites in these organs and tissues, plasma and urine were investigated in both animal genders.  Spirodiclofen ¹⁴C-radiolabelled in the dihydrofuranone ring of the molecule was used for this study.  In Test 1 (male rats, sacrifice 3 hours post dosing) 52% of the dose was detected in the organs and tissues, with quantities in the gastrointestinal tract (44%) and urine (1.8%).  This indicates almost complete absorption and rapid distribution. The percentage of the dose in the body (excluding the gi-tract) declined to 3.3% 24 hours after administration whereas the proportion of residues excreted by urine increased to 58%.  In Test 4 (female rats, sacrifice 3 hours post dosing) 32% of the dose was detected in the organs and tissues, with 53% in the gi-tract and 5.1% in urine. This indicates almost complete absorption and rapid distribution. The percentage of the dose in the body (excluding the gi-tract) declined to 0.3% at 24 hours after  administration, whereas the proportion of residues excreted by urine increased to 75%. With the exception of teh gi-tract, the highest concentrations were detected in the liver. This was generally highest in the organs of animals sacrificed 3 hours after administration and decreased by a factor of >10 to lower than 1% over 3-24 hours post dosing. The values in plasma compared to those in the skin and carcass were about 10 x higher which indicates a delayed equilibrium of the radioactivity between plasma and the tissues. The concentrations in the kidney were more or less comparable to those in the plasma at all time points in both genders.  For elucidation of metabolism, urine and plasma samples, and extracts from liver and kidney were analyzed by HPLC with radiodetection. The main purpose was to  quantify only the major metabolites in the samples i.e. enol, 3-hydroxy-enol (eq. + ax.) and 4-hydroxy-enol (eq. + ax.) because their amount was different in the urine samples of males and females of the first rat metabolism study. The metabolites in urine and plasma were identified by direct LC-MS/MS analysis and for the enol additionally by co-chromatography with the radiolabelled reference compound. The assignment of the metabolites in all HPLC chromatograms was done by comparison either with respective chromatograms of urine samples of the first rat metabolism study or with a mixture of radiolabelled reference compounds taken from the second rat metabolism study The identification rate was high and accounted for most samples of plasma, liver and kidney for more than 90% of the TRR. Only in the 24 hour plasma sample of male rats (TRR = 0.123 mg/kg) the identification rate was 51% that was based on the enol metabolite. The amount of metabolites in the urine samples of both sexes increased over 3-24 hours post dosing. Whereas the enol was dominant in urine samples of female rats, the hydroxylated enol metabolites were the major compounds in the respective samples of male rats.  The amount of the metabolites in plasma samples of both sexes decreased over the same period.  The enol was the by far most dominant metabolite in all samples. Both hydroxylated enol compounds were found only in the plasma of male rats in low levels (<1 % of the dose) and not in the plasma of female rats. This indicated a fast elimination of the compounds from the body via excretion by urine.  The amount of the metabolites in liver of both sexes was decreasing in the same time. Enol was the dominant metabolite in all samples. Both hydroxylated enol compounds were quantitatively different and showed significant higher levels in the livers of male rats. This indicated a higher metabolic activity for further transformation in male rats. The amount of the metabolites in kidney of both sexes was decreasing with time. Enol was again the dominant metabolite in all samples. The higher amounts of the hydroxylated enol compounds in the kidneys of male rats were comparable to those found in the respective urine, plasma and liver samples. Summarising all results, the quantitative differences in the urine spectrum of male and female rats detected in the first rat metabolism study can be explained by higher metabolic cytochrome P450 activity in male rats, fast elimination of the enol and hydroxy-enol metabolites from the body of both sexes via excretion by urine, similar concentrations of the enol in plasma and organs of both sexes at all times.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Absorption

The excretion of radioactivity in urine indicates that after a single low oral dose of 2 mg/kg bw, at least 64% (males) or 76% (females) of spirodiclofen is absorbed within 48 hours. In a further study in which a single oral dose of 2 mg/kg bw was  adminstered to rats, the radioactivity levels in urine and organs demonstrated that at least 60 and 75% (in males and females, respectively) of the administered  dose was absorbed within 24 hours. Urinary excretion over a 168 hour period following a single oral dose of 100 mg/kg bw showed that absorption in male rats was at least 37%. Faecal excretion of radioactivity in bile-duct cannulated rats that were treated with a single oral dose of 1 mg/kg bw suggests incomplete absorption at this dose level. The higher level of faecal excretion after administration of 100 mg/kg bw indicates that the level of absorption decreases with increasing dose level. In a 15-week feeding study, similar absorption values were observed.

The EFSA Conclusion reports oral absorption of 65% for spirodiclofen.  This value is used for risk assessment purposes.

The EFSA Conclusion reports dermal absorption of 0.4% (product concentrate) and 3% (dilution) for spirodiclofen.  A worst case value of 3% is used for risk assessment purposes.

No data are available for the extent of inhalation absorption.  A default assumption of 100% is used for risk assessment purposes, according to REACH Guidance.

Excretion

Excretion of radioactivity was rapid after oral dosing.  After a single administration of 2 mg/kg bw, 58% (males) and 75% (females) of the administered radioactivity was excreted in urine within 24 hours.  At least 88% of the administered dose  (99% of recovered radioactivity) was excreted within 48 hours.  At this dose level, the excretion of radioactivity in expired air was negligible.  In male rats, 168 hours after administration of 100 mg/kg bw, 96% of the administered dose (100% of recovered radioactivity) was excreted. In a 15-week feeding study, similar excretion rates were observed.

Distribution

Levels of radioactivity in organs and tissues were low following oral dosing. Depending on the time of sampling, peak tissue levels were observed at 3 hours after administration (as measured by LSC) or at 4-8 hours after administration (as assessed by autoradiography). At 48 hours after oral administration of 2 mg/kg bw, the highest levels of radioactivity were observed in liver, kidney, plasma,  gastrointestinal tract and skin.  In females, organ and tissue levels were 5-15  times lower than in males.  In male rats treated with spirodiclofen for 15 days, tissue levels were about 4 times lower than in males treated with a single dose of spirodiclofen. Relative distribution of radioactivity was similar for all experimental groups.

Metabolism

There was a marked sex difference in the metabolite profile. Analysis of urine collected over a 48-hour period revealed that in male rats the main urinary metabolites (55-57% of total recovered radioactivity) were the 3- and 4- hydroxy-enol isomers.  In females, the main urinary metabolite (55% of total recovered radioactivity) was the enol metabolite.  In urine collected over a 24-hour period, a similar sex difference in metabolites was found.  In plasma, liver and kidney samples of both male and female rats the main metabolite was the enol.  In these samples the levels of the 3- and 4-hydroxy-enol isomers were higher in males than in females, indicating a higher capacity in the metabolism of the enol metaboite in male rats. The differences in the metabolite profile were quantitative rather than qualitative. Pre-treatment of rats for 15 days or 15 weeks with unlabeled spirodiclofen did not markedly change the metabolism of spirodiclofen. In the faeces of male and female rats treated with 2 mg/kg bw low, levels of the parent compound, enol metabolite and hydroxy-enol isomers were found.  The observation that the MA-cyclohexyl ester and dichlorobenzoic acid are almost exclusively found in faeces suggests that substantial metabolism of spirodiclofen may take place inside the gastrointestinal tract.