thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jan 1995 - 25 Aug 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Reference 2

Reference Type:

other: Amendment No. 1

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 3

Reference Type:

other: Amendment No. 2

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

adopted 1982

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

EC Number:

601-147-9

Cas Number:

111988-49-9

Molecular formula:

C10H9ClN4S

IUPAC Name:

3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

Radiolabelling:

yes

Remarks:

14C-labelled in the positions 4 and 5 of the thiazolidine ring

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann Versuchstierzucht GmbH & Co KG, Borchen, Germany
- Weight at study initiation: approximately 200 g
- Housing: During the non-radioactive pretreatment period, the rats were housed as single animals in plastic cages. During the excretion studies, the animals were kept in special metabolism cages.
- Diet: Altromin 1324 standard food 18 g/day/animal
- Water: tap water, ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
during test period at room temperature and during pretreatment period at 20 °C
- Humidity (%):
during pretreatment period at 40-80%

IN-LIFE DATES: From: 11 Jan 1995 To: 25 Aug 1997

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: low dose: saline solution; high dose: 0.5% Tragacanth

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The administration solution of the low dose experiments was prepared by dissolving the appropriate amounts of labelled compound in saline solution. For the high dose groups the labelled and non-labelled compound were homogeneously mixed in a 0.5% Tragacanth® suspension. Subsequently, an ultrasonic water bath at 70 °C was used for both solutions. The administered volume was 10 mL/kg body weight. The administration was carried out immediately after the preparation of the solution. The compound was stable in this solution for at least 48 h, as tested by high performance liquid chromatography (HPLC).

Duration and frequency of treatment / exposure:

single application

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose / concentration:

5 animals per experiment (in total 15 males and 5 females)

Control animals:

no

Positive control reference chemical:

no

Details on study design:

During the study 4 experiments were conducted:
Experiment 1: A single oral dose of 1 mg/kg bw was administered to 5 male rats for measuring the radioactivity in the expired air.
Experiment 2: The test compound was administered in a single oral dose of 1 mg/kg bw to male rats.
Experiment 3: The test compound was administered in a single oral dose of 1 mg/kg bw to female rats.
Experiment 4: The test compound was administered in a single oral dose of 100 mg/kg bw to male rats.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, feces, expired air, blood (separated into plasma and erythrocytes), organs and tissues and residual carcass
- Time and frequency of sampling:
Urine was sampled in intervals of 0 - 4, 4 - 8, 8 - 24, and 24 - 48 h after administration
Feces was sampled in the intervals 0 - 24 and 24 - 48 h after administration
CO2 was collected separately for each animal in the intervals 0 - 8, 8 - 24 and 24 - 48 h after administration.
-Other:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and feces
- Time and frequency of sampling:
Urine: urine samples of all individual sampling intervals were combined fo each test
Feces: total amount of feces samples collected within the first 48 h
- From how many animals: samples pooled from animals of each test

MEASUREMENT OF RADIOACTIVITY AND USED ANALYTICAL METHODS
The following techniques for measuring radioactivity and for identification of substances were used in the present study:
1) Liquid scintillation counting (LSC) for measuring radioactivity
2) Liquid chromatography–mass spectrometry (LC-MS) combined with a radioactivity detector for measuring radioactivity and for the quantitation of parent compound and/or metabolites in composite urine samples and feces extracts.
3) NMR-spectroscopy for further identification of compounds in most of the samples.

1) LSC
Measurement of liquid samples (urine, plasma, expired air ethanolamine/methanol):
The following liquid scintillation counters used:
LKB 1219 Spectral
Beckman LS 6000

Measurement of solid samples (feces, organs and tissues):
The following liquid scintillation counters used:
Philips PW 4700, Beckman LS 6000 LL

Samples of organs (except fatty organs and tissues) or for an analysis of residues with a low radioactivity content, the samples were weighed and combusted in an oxygen atmosphere using the "Oxidizer 307" (Packard Instruments). "Permafluor E+" (Packard Instruments) was used as scintillator. Fatty organs or tissues were solubilized by means of a tissue solubiliser. Portions of these solutions were filled into scintillation vials together with a suitable scintillation cocktail (e.g. Quickszint 401, Zinsser Analytic GmbH) and the radioactivity was measured in a scintillation counter.

Limit of detection/quantification (LSC):
In addition to the experimental values, background values were determined for all measuring procedures. For this purpose, "blank" samples were prepared for measurement in the same way as the experimental samples. The threshold dose ratio, P limit, is thus defined by a drop in the net count rate (N) to the value of the count rate (blank sample counting rate; NE) that was determined for the blank sample (QF =1). When the sample preparation is readily reproducible a value of 0.33 is used for the quality factor.

2) LC-MS
The following equipment was used:
HPLC: HP1090, HP1090, HP1050
Radioactivity detector: Ramona 5, 6 or 90 (Raytest) with solid scintillator cells
MS: TSQ 7000 instrument by Finnigan

3) NMR-spectroscopy
The following equipment was used:
BRUKER DPX 300, BRUKER DPX 600 and AC 300 instrument by BRUKER (300 MHz)

The sample preparation for quantitation and identification of metabolites:
In general, the identification was conducted by comparative HPLC with authentic reference compounds and also by employing various mass- and NMR-spectroscopic techniques. The quantification of the metabolites was conducted by integrating the 14C-signals in the chromatograms of the native urine samples and the faeces extracts.

Urine
The individuel urine samples of all sampling intervals were combined for each test.

Feces
The total amounts of the feces samples collected within the first 48 h after administration (except aliquots used for radioassay) were combined and intensively mixed. Aliquots of the pooled feces samples were combusted to determine the effective amount of radioactivity in the pooled feces samples. The pooled feces samples were extracted in 15 steps using n-hexane (3 steps), ethanol (1 step), methanol (3 steps) and water (8 steps). The solid residues were dried and aliquots combusted for determination of radioactivity. For identification of compounds, the extracts of the different extraction steps were combined and further processed with a mixture of water, methanol and acetonitrile and a dispersing instrument (Ultraturrax). After repeating this step 3 times, the supernatant samples were combined and their volume reduced. Again methanol was added and the samples were treated with the dispersing instrument. The supernatants were decanted for further analysis. This procedure was repeated four times. The supernatant samples were combined and their volume was reduced prior to HPLC analysis.

Statistics:

Values were checked for outliers by the outlier test of NALIMOV. Values identified as outliers - if any - were marked and not considered in calculations of arithmetic means and standard deviations. Characteristic excretion or residue data from the different animal groups were checked for statistically significant differences using the non-parametric U-test according to Wilcoxon Mann Whitney. Due to the use of different calculation programs, minor differences may occur between these values.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Based on the renal excretion of radioactivity, the absorption was rapid and approximately 75% of the administered dose was absorbed after treatment with the low dose (both sexes) and approximately 60% after treatment with the high dose (males only).

Main ADME results 2

Type:

distribution

Results:

Maximum plasma levels of total radioactivity were reached after 2 to 4 h. 3.2% (males) and 1.6% (females) of the administered radioactivity was measured in organs and tissues mainly liver, kidney, adrenals and thyroid of the low dose test groups.

Main ADME results 3

Type:

excretion

Results:

Elimination was predominantly via the urine in both sexes. The renal and fecal excretion of total radioactivity was 91.3 - 93.4% (low dose) in males and females and 73.5% (high dose) in males only.

Main ADME results 4

Type:

metabolism

Results:

The test item was intensively metabolized. A total of 16 metabolites and the parent compound were identified in the urine and 5 metabolites and the parent compound were identified in feces. Similar metabolite profiles were found for male and female rats.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Based on the renal excretion of radioactivity, the absorption was rapid, Approximately 75% of the administered dose was absorbed after treatment with the low dose (males and females) and 60% after treatment with the high dose (males only). The absorption of radioactivity after oral administration of the test substance was rapid in all tests with t½ (absorption) ranging from 0.17 h to 0.20 h. This was also supported by the absorption lag-times of 0.03 to 0.04 h (low dose) and 0.11 h (high dose). The absorption after administration of the high dose began slightly later than after administration of the low dose. For details, please refer to the attached background material (attachment 1).

Details on distribution in tissues:

Maximum plasma levels of total radioactivity were reached after 2 h to 4 h reaching dose normalized concentrations ranging from P = 0.54 (males, high dose) to P = 0.78 (males, low dose). Also the distribution volumes at steady state (Vss) were 1.45 L/kg bw for males and 1.72 L/kg bw for females, respectively after administration of the low dose. Both values (dose normalized concentrations and distribution volume) demonstrate that the radioactivity of test compound was readily distributed from the blood into tissues and organs of the rats. 48 h after oral administration of the test item, 3.2% (males) and 1.6% (females) of the administered radioactivity was measured in organs and tissues of the low dose test groups. After administration of the low and the high dose, the highest concentrations of radioactivity were measured in liver, kidney, adrenals and thyroid. For details, please refer to the attached background material (attachment 2 and 3).

Transfer into organs

Transfer into organs 1

Key result

Test no.:

#2

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: for males treated with 1 mg/kg bw, radioactivity was readily distributed to the peripheral tissues mainly liver, kidney, adrenals and thyroid.

Transfer into organs 2

Key result

Test no.:

#3

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: for females treated with 1 mg/kg bw, radioactivity was readily distributed to the peripheral tissues mainly liver, kidney, adrenals and thyroid.

Transfer into organs 3

Key result

Test no.:

#4

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: for males treated with 100 mg/kg bw, radioactivity was readily distributed to the peripheral tissues mainly liver, kidney, adrenals and thyroid.

Details on excretion:

Only 0.86% of the administered dose was found in the expired air demonstrating the stability of the position of the radiolabel with respect to formation of volatile substances. The sum of renal and fecal excretion of the total radioactivity 48 h after the administration of the low dose amounted to 91.3% (males) and 93.4% (females) of the administered dose and to 73.5% (males only) for the high dose experiment. The predominant route of excretion was renal. In the low dose group, 76.8% (males) and 82.9% (females) of the administered radioactivity was excreted renally, whereas in the high dose male rats, 60.2% of the administered radioactivity was excreted renally. Residues of the administered radioactivity in the bodies are relatively low for the low dose experiments, but approximately 12.4% of the administered radioactivity remained in the organs and tissues of the rats dosed with 100 mg/kg bw at 48 h after treatment. This included an amount of approximately 4.3% of the administered dose that was measured in the gastrointestinal tract at sacrifice. Thus, indicating an incomplete absorption of total radioactivity after administration of the high dose. The accumulated renal excretion, however, did not reach a plateau during the test period of 48 h, demonstrating that the amounts of radioactivity measured in the body at sacrifice is subject to further excretion. For details, please refer to the attached background material (attachment 1).

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

AUC: low dose: 9.19 µg/mL*h (males) and 10.4 µg/mL*h (females); high dose: 1560 µg/mL*h (males only)

Toxicokinetic parameters 2

Key result

Toxicokinetic parameters:

half-life 1st: low dose: 2.2 h (males) and 3.3 h (females); high dose: 4.0 h (males only)

Toxicokinetic parameters 3

Key result

Toxicokinetic parameters:

half-life 2nd: low dose: 19.0 h (males) and 44.5 h (females); high dose: 9.9 h (males only)

Toxicokinetic parameters 4

Key result

Toxicokinetic parameters:

Cmax: low dose: 2.0 h (males) and 3.0 h (females); high dose: 4.0 h (males only)

Toxicokinetic parameters 5

Key result

Toxicokinetic parameters:

Tmax: low dose: 1.61 h (males) and 3.04 h (females); high dose: 9.82 h (males only)

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Following oral administration, the test substance was intensively metabolized in male and female rats. 16 metabolites and the parent compound were identified after isolation from urine pool, whereas 5 metabolites were identified in feces (beside the parent compound). All metabolites identified in the feces were also found in the urine. The main metabolite in the urine of the animals treated with the low dose was N-[(cyanoamino)(methylthio)methylene]-glycine (PIZ 1250) in males, amounted to approximately 10% on average of the administered radioactivity and sulfuric acid mono-[5-(2-cyanoimino-thiazol-3-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethyl] ester (PIZ 1243) in females amounted to approximately 22% on average of the administered radioactivity. After administration of the high dose, the major metabolite was 3-(6-chloro-pyridin-3-ylmethyl)-4-hydroxy-thiazolidine-2-ylidenecyanamide (PIZ 1265), which was also conjugated with glucuronic acid (PIZ 1270). The parent compound only accounted for 2.3% to 3.7% of the administered dose in the urine pools of all tests. These low amounts demonstrate that the test compound was intensively metabolized by the rats after oral administration. For details, please refer to the attached background material (attachment 5 and 6).

Enzymatic activity

Enzymatic activity measured:

not measured

Applicant's summary and conclusion

Conclusions:

The toxicokinetic behavior and metabolism of the test compound was investigated in a GLP-compliant study according to EPA OPP 85-1 guideline. The study is therefore considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. During the study, 14C-labelled test compound was administered in a single oral dose of 1 mg/kg bw to male and female rats and additionally in a single oral dose of 100 mg/kg bw to male rats. The present study demonstrated that the test compound was rapidly absorbed and readily distributed to the peripheral tissues, mainly the liver, kidney, adrenals and thyroid. There was no evidence of accumulation of the radioactivity in the organs or tissues. Excretion of the low dose was almost complete, occurring mainly via the urine and to a lesser extend via feces. Approximately 4.3% of the administered high dose radioactivity was measured in the gastrointestinal tract at sacrifice, indicating an incomplete absorption of the total radioactivity after treatment with the high dose. The test substance was intensively metabolized, as 16 metabolites and the parent compound were identified after isolation from urine pool, whereas 5 metabolites and the parent compound were identified in feces. All metabolites identified in the feces were also found in the urine.