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

Basic toxicokinetics

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

Cas Number:

148477-71-8

Molecular formula:

C21H24Cl2O4

IUPAC Name:

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

Radiolabelling:

yes

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

single high dose experiment

Doses / concentrations 2

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

single low dose experiment;
CO2-test

Doses / concentrations 3

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

single low dose experiment;
EPA-basic test

Doses / concentrations 4

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

single low dose experiment

Doses / concentrations 5

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

multiple dosing test,
low dose

Doses / concentrations 6

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.

Results and discussion

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.

Main ADME results

Main ADME results 1

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.

Main ADME results 2

Type:

excretion

Results:

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

Main ADME results 3

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.

Main ADME results 4

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)

Toxicokinetic / pharmacokinetic studies

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).

Transfer into organs

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.

Metabolite characterisation studies

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.