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

Endpoint summary

Link to relevant study record(s)

Reference

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

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

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

Radiolabelling:

yes

Remarks:

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

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

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

Dose / conc.:

2.5 mg/kg bw/day

Dose / conc.:

5 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

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".

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.

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.

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.

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.

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).

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).

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])

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])

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])

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])

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])

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 measured:

not measured

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.

Description of key information

Absorption of the test substance is to be taken into consideration after oral exposure, whereas dermal and respiratory absorption are rather unlikely. In fact, with respect to the skin, the high water solubility and poor lipophilicity of the test substance, as well as the absence of a dermal irritating and/or toxic potential indicate that dermal absorption is not to be expected. Due to the low vapour pressure of the test substance, the moderate to high solubility in water and the non-toxicity after acute inhalation exposure, respiratory absorption is considered unlikely although the test substance has a moderate log Pow value. With respect to the oral route, biokinetic studies showed that the test substance is rapidly and almost completely absorbed from the intestinal lumen. Following absorption, the test substance is rapidly distributed to different organs and tissues. The metabolisation rate of the test substance in rodents is moderate and the elimination from the organism is fast and complete. There is no indication of any bioaccumulation potential of the parent compound and/or its metabolites.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Additional information

Study summaries

The toxicokinetic and metabolic behavior of the test substance was investigated in the rat in a GLP-compliant study according to the OECD Guideline 417 and was regarded as key study (M-024723-02-1). During the study, 8 treatment groups (basically 4 animals per sex) were orally treated with different doses of two 14C labelled compounds denotes [nitroimino-14C]-label and [thiazolyl-2-14C]-label in the range of 2.5 to 250 mg/kg bw. During intervals of maximal 72 hours after treatment, expired air, urine, feces, carcass, blood, organs and tissues were collected. Radioactivity of the samples were analyzed by liquid scintillation counting (LSC). Urine and fecal samples were further analyzed by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC, urine only) with radiodetection for metabolite identification. In addition, on one treatment group, whole body autoradiography (radioluminography) was performed between 1 and 72 hours after treatment.

Absorption:

The test substance was rapidly and extensively absorbed from the gastrointestinal tract. In fact, 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 of male and female rats. 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.

Distribution:

Plasma concentrations of radioactivity show peak values between 1.27 – 1.82 mg equivalents/mL after 1.5 hours following oral administration of 2.5 mg/kg bw. 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. Further, radioactivity was rapidly distributed to all tissues within one hour after 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% of administered dose for all dose levels and dose regimens. Therefore, there was no sign of accumulation in any one of the organs or tissues.

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 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% of 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% of 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. 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.

Metabolism:

The predominant component identified for all doses and dose regimens was the parent compound, which accounted for between 55 and 74% of administered dose. The major metabolites identified, accounting for >10% 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 nitroguanidine (NTG) at 1.42 – 3.92% of 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% of administered dose and together accounted for less than 11% of 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).

In total, 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 is also 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, TZNG and MNG. Except for a saturated absorption and transiently delayed elimination noticed at higher dose, the biokinetics and metabolism of the test item were not markedly influenced by dose level, dose regimen and sex.

 

A further study conducted in mice is available, which has been considered for support. In this study, the biokinetic behavior of the test substance was investigated in mice, according to USA EPA OPPTS 870.7485 (M-089752-01-1). In fact, 5 male and 5 female mice were orally treated with a single dose of 5 mg/kg bw of [nitroimino-14C]-labelled test compound. Urine and feces were collected over a 24 hour interval (from 5 animals per sex), whereas organs and tissues were sampled 7 days after treatment (from 3 animals per sex). Radioactivity was measured by LSC and metabolites were identified using two-dimensional TLC and LSC.

Absorption:

After a single oral administration of the radioactive-labelled compound at a dose of 5 mg/kg bw to male and female mice, most of the administered radioactivity (% dose: 99.2% for males - 98.7% for females) was excreted into urine and feces within 7 days after the administration. Based on the amount of radioactivity excreted via urine, absorption was >92%.

Distribution:

The concentration of 14C-labelled compound in the liver of both male and female mice, in adrenal and blood of male mice, and in hair of female mice was 0.02 µg equiv/g. Less than 0.01 µg equiv/g were found in all other tissues of male and female mice. The percentage of 14C in all tissues was in a range of <0.01 to 0.02% of the initial administered dose.

Therefore, it was considered that most of the orally administered compound was absorbed into the body with no tendency of persistence or accumulation in any specific tissue.

Excretion:

Urinary excretion rates (92.4% - 93.7% of administered dose) within 7 days after administration were markedly higher than the fecal excretion rates (6.8% - 5.0% of administered dose) for both sexes. Thus, indicating that urine was the main excretion route of administered test item.

Metabolism

Unchanged parent compound was the major radioactive component recovered in urine and feces. The major metabolite in urine and feces was TZNG. MNG and NTG were also present in urine and feces, with TMG and 3 other minor components present in feces only. The results of the metabolite characterization showed common metabolites for both male and female mice after single oral application of 5 mg/kg bw. The metabolism of the test compound in mice was thought to involve (i) demethylation leading to TZNG and cleavage of the nitrogen-carbon bond between nitroguanidine and the thiazolylmethyl group of TZNG to yield NTG; (ii) cleavage of the nitrogen-carbon bond between nitroguanidine and the thiazolylmethyl group leading to MNG and demthylation of MNG to yield NTG and (iii) denitration of the test substance leading to TMG.

Overall, the present study demonstrated that the test compound is rapidly absorbed and excreted in male and female mice. Furthermore, no evidence of accumulation in organs or tissues were found. Beside the parent compound, the major metabolites detected in urine, but to a considerable lesser extent also in feces were TZNG, NTG and MNG, whereas TMG and 3 other minor components were present in feces only. Therefore, the results of this supporting study carried out in mice are in line with the results of the above-mentioned key study conducted with rats.

 

An in vitro study on the comparative metabolism of the [14C]-labelled test compound is available, which was conducted for inter-species comparison (M-504338-02-1). The study will only be shortly mentioned here, for purpose of data completeness. For investigating the comparative metabolism, the test substance was incubated with liver microsomes from male Wistar rats and humans in the presence of NADPH cofactor. The test compound was incubated for 1 hour at a concentration of 10 µM, with a microsomal protein concentration of 1 mg/mL. The sampling times were 0, 0.5 and 1 hour after test start. Samples were analyzed following protein precipitation by reversed phase HPLC with radiochemical detection (HPLC-RAD). The metabolic activity of the microsomes was demonstrated by determining 6β-hydroxytestosterone that was formed from testosterone by testosterone 6β-hydroxylase.

The recovery of radioactivity was ≥85%. No metabolites were detected in human liver microsome incubations. A single degradation product was present in the control and 1 hour sample for rat liver microsomes but at concentrations below the lower limit of quantification. It can be concluded that liver microsomal phase I metabolism enzymes are unlikely to play a significant role in the biotransformation of the test substance in the rat or human.

 

Conclusion and assessment of the toxicokinetic behavior of the test substance

 In accordance with Regulation (EC) 1907/2006, Annex VIII, Column 1, Item 8.8 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), assessment of the toxicokinetic behavior of the test substance was conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physicochemical properties. The test substance is a solid white to yellow colored powder with a water solubility of 327 mg/L at 20 °C, a molecular weight of 249.68 g/mol and a vapour pressure 1.3x10^-10 Pa at 25 °C. The octanol/water partition coefficient (log Pow) was determined around 0.7 at 25 °C.

Absorption

In general, absorption of a substance depends on the potential to cross biological membranes, which is determined by the molecular weight, the log Pow and water solubility. Mostly, substances cross the membranes by passive diffusion, which requires sufficient solubility in water and lipids, a capability, which is described by the log Pow. In general, log Pow values between -1 and 4 are favorable for absorption whereas ionic substances are thought not to readily diffuse across biological membranes. Chemicals that do not offer these properties may be absorbed via active processes including facilitated diffusion, active transport or pinocytosis (ECHA, 2017).

Oral:

In general, molecular weights below 500 and log Pow values between -1 and 4 are favorable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (>1 mg/L). As the test substance has a molecular weight of 249.68 g/mol, a water solubility of 327 mg/L (at 20 °C) and a log Pow value of 0.7 (at 25 °C), systemic exposure by oral absorption is likely. This is also supported by systemic toxicity, which occurred after oral administration of the test substance. Furthermore, mortalities were observed in mice and rats after single oral administration of the test substance, which provide evidence that absorption has occurred. In addition, biokinetic studies in rats showed that the test compound is rapidly and almost completely absorbed from the intestinal lumen. Therefore, the test substance is considered to be absorbed along the gastrointestinal tract.

Dermal:

To enable dermal absorption, the substance first has to penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network (ECHA, 2017). The stratum corneum provides the first barrier against hydrophilic compounds and dermal uptake of substances with poor lipophilicity (log Pow <0) will be impeded. Log Pow values <–1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, and hence dermal absorption is likely to be low. In general, if water solubility is above 10000 mg/L and the log P value is below 0 the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Dermal uptake for such a substance will be low (ECHA, 2017). Since the test substance has a water solubility of 327 mg/L, absorption is anticipated to be moderate to high. However, the log Pow of the test substance is 0.7 and this rather poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. According to the ECHA, log P values between 1 and 4 favor dermal absorption (values between 2 and 3 are optimal) particularly if water solubility is high. In addition, as the test substance is a solid, hindered dermal absorption has to be considered, as dry particulates first have to dissolve into the surface moisture of the skin before uptake via the skin is possible (ECHA, 2014). Furthermore, data on acute (M-027396-01-1) and subacute dermal toxicity (M-027480-01-1) of the test substance are available. In both studies, the test compound was proved to be non-toxic after dermal application to rats, which also supports the conclusion that dermal absorption of the test compound is low, especially since single oral application of the test substance led to systemic effects and mortalities in rodents. Moreover, an in vivo skin irritation study on rabbits (M-027402-01-1) revealed that the test compound is not irritating to the skin and hence, facilitated penetration due to local skin damage can be excluded.

Overall, the high water solubility, poor lipophilicity, the non-toxicity after dermal exposure and the non-irritating properties to the skin indicate that dermal absorption of the test substance is rather unlikely.

Inhalation:

Substances including gases, vapours, liquid aerosols (both liquid substances and solid substances in solution) and finely divided powders/dusts may be absorbed directly from the respiratory tract or, through the action of clearance mechanisms, may be transported out of the respiratory tract and subsequently be swallowed which might lead to absorption in the gastrointestinal tract (ECHA, 2017). In general, substances with a low vapour pressure of <500 Pa are not favorable for respiratory absorption as those substances are not available for inhalation as vapour (ECHA, 2017). The test substance has a low vapour pressure of 1.3x10^-10 Pa at 25 °C and thus being of low volatility. Moderate log Pow values (between -1 and 4), such as noted for the test substance, are favorable for absorption directly across the respiratory tract epithelium by passive diffusion. However, the test substance is moderate to high soluble in water and passive transfer through cell membranes in the respiratory tract will be impeded. Therefore, resorption of the test substance following inhalation is not expected to be significant under normal use and handling. However, inhalation of aerosols cannot be excluded. With regards to inhalation toxicity, an acute toxicity study in rats is available (M-027390-01-1). This study revealed that the test substance possesses no acute toxicity on uptake once via the respiratory tract in the form as dust (relatively low inhalability). Therefore, the available data on inhalation toxicity indicate that respiratory absorption is unlikely.

Overall, due to the low vapour pressure, respiratory absorption following inhalation is not considered as significant. This is supported by the results of the acute inhalation toxicity study, in which no signs of systemic toxicity was observed in rats after inhalation exposure.

Distribution

In the rat and mouse, the test substance was widely and homogeneously distributed over various organs (tmax=1.5 hours), with rapid decrease of residues to levels near or at LOQ at 72 hours. There was no evidence of accumulation, although up to 4 hours post-dosing, higher levels were detected in urinary bladder, kidney and liver reflecting the role of these organs for excretion and metabolism.

Excretion

At 24 hours after dosing to rats and mice, about 95% of the radioactivity was excreted. The urinary excretion was the dominating route of elimination. The excretion profile after high-dose administration to rats was almost identical to that after low-dose administration, although the plasma concentration exhibited a bi-phasic kinetics, with concentration peaks at 6 hours and 32 hours, suggesting a moderate enterohepatic cycling.

Metabolism

The metabolisation rate of the test substance in the rat was moderate, as the test substance was excreted unchanged at 56 - 74% 72 hours after administration. The main metabolic pathway was (i) oxidative demethylation, and (ii) cleavage of the nitrogen-carbon bond between the thiazolyl-methyl position and the nitroimino moiety. The main urinary metabolites, recovered after low-dose testing were TZNG (7 - 11%), MNG (8 - 13%) and NTG (1 - 4%). In the feces, MTCA (9%) and TMG (2%) were recovered. Other characterized metabolites were present at <2% of administered dose. In the mouse, the test substance was metabolized slightly faster than in the rat but the route of metabolism was similar. Unchanged parent compound accounted for approximately 40% of the dose. The major urinary metabolites were TZNG (approximately 3 %), MNG (9%), NTG (11%). The urinary metabolites were also seen in the feces (0.2 - 1.3%), as was TMG (1%) and 3 unidentified metabolites which accounted for <1% of the dose.

 

References not included in IUC:

M-504338-02-1, Solà, J. 2014: [Thiazolyl-(methyl)-14C]Clothianidin: Metabolic Stability and Profiling in Liver Microsomes from Rats and Humans for Inter-Species Comparison, Testing laboratory: Harlan Laboratories S.A, Barcelona, Spain, Report no.: EnSa-14-0237, Owner company; Bayer AG, Monheim, Germany, Study number: S48275, Report date: Nov 21, 2014