clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Sep 1997- 11 Oct 2000

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)

Version / remarks:

assumed 1984 since the study was conducted 2000

Deviations:

yes

Remarks:

animal housing conditions slightly different compared to the actual guideline version (2010), rationale for selection of dose and vehicle not provided

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

433-460-1

EC Name:

-

Cas Number:

210880-92-5

Molecular formula:

C6H8ClN5O2S

IUPAC Name:

(E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine

Radiolabelling:

yes

Remarks:

Two 14C labelled compounds were used: denotes [nitroimino-14C]-label denotes [thiazolyl-2-14C]-label

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: males: about 210 g; females: about 210 g
- Housing: During the acclimation the rats were housed in plastic cages on wood shavings. After administration of the radiolabelled test compound, the rats were kept individually in Makrolon® metabolism cages.
- Diet: Altromin 1324 standard feed for rats, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: about 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 27 - 65
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 16 Sep 1997 To: 12 Aug 2000

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

other: 0.5% aqueous Tragacanth solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Non-labelled compound was weighted into a glass flask, an adequate portion of the stock solution of radiolabelled compound was pipetted into the same flask and blown dry under a gentle stream of nitrogen. The dry residue was reconstituted in a 0.5% aqueous Tragacanth solution and the suspension homogenised by ultrasonication in a water bath at ca. +70 °C for about 20 min. The suspension was stirred overnight at room temperature. Before administration the suspension was heated in a water bath at ca. + 70 °C for about 10 min. The administration solutions were prepared one day before the administration.

Duration and frequency of treatment / exposure:

single application

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.5 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose / concentration:

basically, 4 animals/sex
For details, please refer to table 1 under "Any other information on materials and methods incl. tables".

Control animals:

no

Positive control reference chemical:

no

Details on study design:

For details, please refer to table 1 under "Any other information on materials and methods incl. tables".

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 erythocytes), organs and tissues and residual carcass
- Time and frequency of sampling:
Expired air was sampled in intervals of 0-4, 4-8, 8-24, 24-48, 48-72 hours.
Urine was sampled in intervals of 0-4, 4-8, 8-24, 24-48, 48-72 hours.
Feces was sampled in periods of 0-4, (4-8), 8-24, 24-48, 48-72 hours.
Carcass, organs and tissues were sampled 72 hours prost treatment.

- Other: One group of six male rats was used for whole body autoradiography (radioluminography). Therefore, one animal was anaesthetised by CO2 inhalation and killed by exsanguination after 1, 4, 8, 24, 48 and 72 hours post-treatment, respectively.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and feces
- Time and frequency of sampling:
Urine was sampled in intervals of 0-4, 4-8, 8-24 hours.
Feces was sampled in periods of 0-24, 0-48, 0-72 hours.
- From how many animals: Samples were pooled for the given time ranges within one test group

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) For the measurement of radioactivity liquid scintillation counting (LSC) was used.
2) For the measurement of radioactivity in whole-body sections of animals radioluminography was used.
3) For separation and evaluation of compounds High Performance Liquid Chromatography (HPLC) and thin layer chromatography (TLC) with radiodetection was used
4) For identification of compounds Mass Spectroscopy was used (HPLC/MS/MS)

1) LSC
Measurement of liquid samples:
The following liquid scintillation counters used:
LKB/Wallac 1219 Spectral
Beckman LS 6000 LL
Beckman LS 6500.

Measurement of solid samples:
Samples were weighed and combusted in an oxygen atmosphere using either the "Oxidiser 307" (Packard Instruments) or the "OX 500" (Harvey Instruments Corporation). Carbosorb was used to trap the combustion product carbon dioxide. Permafluor E+ (Packard Instruments) was used as scintillator. For analysis with the "OX 500", Oxysolve C-400 (Zinsser Analytic GmbH) was used for absorption and as scintillation cocktail. Test materials for combustion with the Oxidizer 307/OX 500 were feces, erythrocytes, organs and tissues like e.g. spleen, liver, lung, bone, muscle, gastrointestinal tract (GIT), solids (e.g. from feces after extraction).
Small organs or tissues were solubilised by means of a tissue solubiliser (e.g. BTS-450, Beckman). Portions of these solutions were filled into scintillation vials together with a suitable scintillation cocktail (e.g. Quickszint401, Zinsser Analytic GmbH). The radioactivity was measured in a scintillation counter. These method was used for the following test materials: adrenal glands, thyroid, ovaries, renal fat and uterus.
The following liquid scintillation counters used:
Philips PW 4700
Beckman LS 6000 LL
LKB/Wallac 1219 Spectral

Limit of detection/quantification (LSC):
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. The limit of detection (LOD) was established as 10 dpm/aliquot after correction for background radioactivity and the LOQ was established as 2 to 3 times the background radioactivity (dpm) of each instrument/method.

2) Whole-body Autoradiography (Radioluminography)
Sections were cut at 50 mm, freeze-dried, imaging plates applied and exposed for between 8 and 96 hours. After exposure the imaging plates were scanned using the Fuji BAS 5000 image analyser and the resulting pictures printed on a colour printer (Fuji Pictography High Grade). In order to correct for possible chemographic effects, a single animal was orally administered with the same dose of the non-radioactive compound, sacrificed after 4 hours and the sections were exposed under identical conditions as described above. The radioactivity was quantified using the software "Tina®" (Raytest, version 2.10g).

Limit of detection/quantification (Radioluminography):
The limit of detection (dpm/g) was established as the 2-fold background radioactivity (PSL/mm2) multiplied with the calibration factor CF derived from Amersham microstandards. The limit of quantitation (LOQ) was fixed as the higher data of a) the 2 fold of the respective LOD-value or b) the lowest value of the calibration microstandards that was included in the calibration curve, whichever was higher.

3) HPLC and TLC
Urine samples and fecal extracts were analysed by HPLC and TLC (urine only) with radiodetection. The chromatograms were recorded and evaluated using the dedicated software package GINA by Raytest (Nuclear Interface, D-48147 Munster, Germany). Reversed Phase HPLC methods of three different selectivities were used in this study.
- acidic eluent: water + % formic acid or acetic acid/acetonitrile gradients
- neutral eluent: water + acetic acid + ammonium acetate/acetonitrile gradients
- ion pair eluent water + heptane sulfonic acid/methanol + water + heptane sulfonic acid gradients
Two TLC systems of different selectivity on amino plates and silica plates were used to separate the polar fraction of metabolites after pre-fractionation with HPLC. All major metabolites were quantified and then identified by co-chromatography with reference compounds. Unresolved, co-eluted polar fractions were further analysed by TLC.

Limit of detection/quantification (HPLC):
LOD was established as a signal to noise ratio of at least 2.5.

4) HPLC/MS/MS
The electro-spray ionisation MS spectra (ESI) were obtained with a TSQ 7000 instrument by Finnigan. Sheath gas pressure: 70 psi, capillary temperature: 270 °C. For the MS/MS experiments, argon was used as the collision gas (pressure in the collision chamber: 2.7 mT). A radioactivity detector (Ramona 90, Raytest) was coupled via a flow splitter between HPLC instrument (HP 1050, Hewlett Packard) and mass spectrometer.

Statistics:

Statistical evaluation and calculations were produced with computer assistance (Hewlett Packard HP3000). The dedicated software package ISOLAB generated for the study protocol controlled online data acquisition and subsequent evaluation was used. Prior to the listing, the values were checked for outliers in accordance with the outlier test by Nalimov, if appropriate. Values identified as outlier were marked and not taken in account in calculations of arithmetic means
and coefficients of variations. Also, the concentrations or amounts of radioactivity were checked for values below the limit of quantitation (LOQ). Values below the LOQ were not considered in calculations of arithmetic means.
For additional details, please refer to "Any other information on materials and methods incl. tables".

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The test substance was rapidly and extensively absorbed from the gastrointestinal tract. Based on the extent of cumulative urinary elimination, the extent of oral absorption was >89% for all doses and dose regimens, for both 14C-labels and sexes.

Main ADME results 2

Type:

distribution

Results:

Maximum plasma levels were measured after 1.5 h (2.5 mg/kg bw), 4 h (25 mg/kg bw) and 6 h (250 mg/kg bw). Radioactivity was distributed rapidly to the organs/tissues mainly nasal mucosa, kidney, liver and urine bladder.

Main ADME results 3

Type:

excretion

Results:

First phase elimination half-life for all groups was within the range of 0.88 – 1.9 hours. Elimination was rapid and predominantly via the urine (89.1 – 94.6% of administered dose) compared to a fecal elimination of 3.3 – 8.6% of administered dose.

Main ADME results 4

Type:

metabolism

Results:

The predominant component identified for all doses and dose regimens was the parent compound, which accounted for between 55 - 74% of administered dose. The major metabolites identified, accounting for >10% of administered dose were TZNG and MNG.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Single doses of 2.5 or 250 mg/kg bw, and repeated doses of 25 mg/kg bw were rapidly and extensively absorbed from the gastrointestinal tract. An average of more than 90% of the administered test substance was absorbed after oral administration in the dose range between 2.5 mg/kg bw and 250 mg/kg bw. No significant difference was observed between the two 14C-labels with regards to absorption. Calculated absorption half-life for all groups was within the range 0.004 – 0.51 hours. Similarly, absorption lag times for all groups were very short and within the range 0.05 - 0.08 hours, although absorption half-life and lag time could not be calculated for the 250 mg/kg dose bw.
For details, please refer to the attached background material (attachment 1).

Details on distribution in tissues:

Plasma concentrations of radioactivity show peak values between 1.27 – 1.82 mg equivalents/mL 1.5 hours after oral administration of 2.5 mg/kg bw in test 2, test 3 and test 7. The maximum plasma concentration following treatment at 250 mg/kg bw was approximately 79.5 mg equivalents/g, which occurred 6 hours after administration. However, at this high dose level a further peak concentration of 47.8 mg equivalents/g occurred at 32 hours, indicating saturated absorption. Due to non-linear absorption kinetics, pharmacokinetic parameters could not be calculated for this dose level. Further, radioactivity was rapidly distributed to all tissues within one hour of oral administration of 5 mg/kg bw, at which time most tissues showed their maximum concentration. Highest concentrations of radioactivity were measured in the urinary bladder, kidney, liver and in the nasal mucosa. Residual radioactivity in the carcass at 72 hours post-treatment was in the range 0.093 – 0.327% administered dose for all dose levels and dose regimens. Therefore, there was no sign of accumulation in any one of the organs or tissues.
For details, please refer to the attached background material (attachment 2 and 3).

Transfer into organs

Key result

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was distributed rapidly to the peripheral tissues mainly urinary bladder, kidney, liver and in the nasal mucosa.

Details on excretion:

Elimination was rapid and predominantly via the urine (89.1 – 94.6% of administered dose) compared with fecal elimination of 3.3 – 8.6% of administered dose. The extent of urinary elimination in males was not influenced by dose level, dose regimen or position of the 14C-label, since cumulative elimination in these groups was within the range 89.1 – 93.0% of administered dose. Females treated at 2.5 mg/kg bw showed a marginally higher cumulative urinary elimination (94.6%) than the male groups.
Mean residence times (MRT) for a 3-compartment model were calculated to be in the range 7.7 – 13.3 hours. First phase elimination half-life (elimination t1/2 [1]) for all groups was within the range 0.88 – 1.9 hours, indicating rapid elimination.
Both urinary and fecal elimination was almost complete within 24 hours of administration for the 2.5 mg/kg bw dose level and for repeated doses of 25 mg/kg bw (94.2 – 97.5% administered dose). However, elimination of the 250 mg/kg bw dose was transiently retarded at 24 hours (60.7% of administered dose). Nevertheless, 72 hours after treatment, total urinary and fecal elimination was within the range 95.4 – 99.6% administered dose for all dose levels and dose regimens. The expiration of 14CO2 and other 14C-labelled volatiles was tested following a single oral administration of 2.5 mg/kg bw in a male rat. During the test period of 72 hours, only 0.017% of the administered radioactivity was expired. This demonstrates the high stability of the labelling position in view of a possible formation of volatile products.
For details, please refer to the attached background material (attachment 1).

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Toxicokinetic parameters:

AUC: 10.3 µg/mL hour (2.5 mg/kg bw male [nitroimino-14C]), 7.28 µg/mL hour (2.5 mg/kg bw female [nitroimino-14C]), 116 µg/mL hour (25 mg/kg bw male [nitroimino-14C]), 10.2 µg/mL hour (2.5 mg/kg bw male [thiazolyl-2-14C])

Toxicokinetic parameters 2

Key result

Toxicokinetic parameters:

half-life 1st: 1.2 hours (2.5 mg/kg bw male [nitroimino-14C]), 1.49 hours (2.5 mg/kg bw female [nitroimino-14C]), 1.89 hours (25 mg/kg bw male [nitroimino-14C]), 0.882 hours (2.5 mg/kg bw male [thiazolyl-2-14C])

Toxicokinetic parameters 3

Key result

Toxicokinetic parameters:

half-life 2nd: 54.1 hours (2.5 mg/kg bw male [nitroimino-14C]), 22.6 hours (2.5 mg/kg bw female [nitroimino-14C]), 28.3 hours (25 mg/kg bw male [nitroimino-14C]), 37.0 hours (2.5 mg/kg bw male [thiazolyl-2-14C])

Toxicokinetic parameters 4

Key result

Toxicokinetic parameters:

Cmax: 1.82 µg/mL (2.5 mg/kg bw male [nitroimino-14C]), 1.29 µg/mL (2.5 mg/kg bw female [nitroimino-14C]), 15 µg/mL hour (25 mg/kg bw male [nitroimino-14C]), 1.27 µg/mL (2.5 mg/kg bw male [thiazolyl-2-14C])

Toxicokinetic parameters 5

Key result

Toxicokinetic parameters:

Tmax: 1.5 hours (2.5 mg/kg bw male [nitroimino-14C]), 1.35 hours (2.5 mg/kg bw female [nitroimino-14C]), 2.70 hours (25 mg/kg bw male [nitroimino-14C]), 2.08 hours (2.5 mg/kg bw male [thiazolyl-2-14C])

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The predominant component identified for all doses and dose regimens was the parent compound, which accounted for 55 to 74% of administered dose. The major metabolites identified, accounting for >10% of administered dose were N-(2-chlorothiazol-5-ylmethyl)-N’-nitroguanidine (TZNG) and N-methyl-N’-nitroguanidine (MNG). 2-methylthiothiazole-5-carboxylic acid (MTCA) occurred at 8.54% and NTG (nitroguanidine) at 1.42 – 3.92% administered dose. These metabolites occurred predominantly in urine.
The main metabolite occurring in feces was N-(2-chlorothiazol-5-ylmethyl)-N’-methylguanidine (TMG). All other metabolites occurred at less than 2% administered dose and together accounted for less than 11% administered dose.
The main routes of metabolism are oxidative demethylation to form TZNG and cleavage of the nitrogencarbon bond between the thiazolylmethyl group and the nitroguanidine moiety. The part of the molecule traced by the [nitroimino-14C]-label is metabolized mainly to MNG and NTG, whereas the thiazol moiety is metabolized to 2-chlorothiazole-5-carboxylic acid (CTCA) and further to MTCA. Other minor routes metabolized the nitroimino moiety to the urea derivatives N-(2-chlorothiazol-5-ylmethyl)-N’-methylurea (TZMU) and 2-chlorothiazol-5-ylmethylurea (TZU), the guanidines TMG and 2-chlorothiazol-5-ylmethylguanidine (TZG) and to N-2-chlorothiazol-5-ylmethyl-N-hydroxy-N’-methyl-N’’-nitroguanidine (THMN). For the proposed metabolic pathway, please refer to the attached background material (attachment 6 and 7).

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 OECD 417. During the study, rats were orally treated with 2.5, 5.0, 25 or 250 mg/kg bw of the test subtance. The study is considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. With the use of radioactive-labelled test material, the present study demonstrated that the test compound is rapidly absorbed and distributed to the peripheral tissues, mainly the liver, kidney, urine bladder and nasal mucosa. The radioactivity was rapidly and almost completely eliminated from all tissues with no evidence of accumulation. In addition, excretion was fast, occurring mainly via the urine and to a lesser extend via feces. However, high doses of the test substance were found to saturate the gastrointestinal absorptive mechanism, resulting in transient delay in excretion. Two major metabolites are formed in vivo, accounting for >10% of administered dose, N-(2-chlorothiazol-5-ylmethyl)-N’-nitroguanidine (TZNG) and N-methyl-N’-nitroguanidine (MNG). Except for saturated absorption and transiently delayed elimination at higher dose levels, the biokinetics and metabolism of test item were not markedly influenced by dose level, dose regimen and sex.