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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

adopted 1982

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

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

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

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

Dose / conc.:

1 mg/kg bw/day

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.

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).

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.

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.

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.

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).

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.

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.

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).

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)

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)

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)

Key result

Toxicokinetic parameters:

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

Key result

Toxicokinetic parameters:

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

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 measured:

not measured

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.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jan 1994 - 31 Oct 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

adopted 1982

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-labelled compound denoted [methylene-14C]-label

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: 15 Jan 1991 To: 19 Oct 1994

Route of administration:

other: oral and intravenous

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-radiolabelled 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 bw for the oral doses and 5 mL/kg bw in case of intravenous dosing. 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 dose exposure and repeated-dose exposure for 14 days (for details, please refer to "Details on study design")

Dose / conc.:

1 mg/kg bw/day

Remarks:

oral

Dose / conc.:

100 mg/kg bw/day

Remarks:

oral

Dose / conc.:

1 mg/kg bw/day

Remarks:

intravenous

No. of animals per sex per dose / concentration:

5/sex/condition

Control animals:

no

Positive control reference chemical:

no

Details on study design:

For duration and frequency of treatment, rats were allocated to 4 groups as follows:
Group A (experiment 1): single oral dose of 1 mg/kg bw (for expiration test), males and females
Group B (experiment 2): single intravenous dose of 1 mg/kg bw, males and females
Group C (experiment 3): repeated-doses of 1 mg/kg bw non-labelled compound once daily for 14 days followed by 1 mg/kg bw of a single oral radioactive dose after 24 h, males and females
Group D (experiment 4): single oral dose of 100 mg/kg bw, males and females

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.
Organs and tissues: directly after sacrifice

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and feces
- Time and frequency of sampling:
Urine: urine samples of the male and female high dose groups were combined and used for the isolation and identification of renal metabolites
Feces: Lyophilized feces of the sampling intervals 0 - 24 h and 24 - 48 h after treatment of all animals were combined within each test group
- 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) Gas chromatography–mass spectrometry (GC-MS)
4) 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:
Beckman LS 7800; Philips PW4700; LKB Spectral 1219

Measurement of solid samples (feces, organs and tissues):
The following liquid scintillation counters used:
Beckman LS 7800; Philips PW 4700

Samples of organs with weights below 500 mg or for an analysis of residues with a low radioactivity content, the samples were weighed and combusted in an oxygen atmosphere using the "Oxidizer 306" (Packard Instruments) to combust samples with weights up to 500 mg an "Ox 300" (Harvey Instruments
Corporation) was used. As scintillator "Permafluor V" (Packard Instruments) was used. Fatty organs or tissues were solubilized by means of a tissue solubilizer. 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: HP1050
Radioactivity detector: Ramona 4 or 6 (Raytest) with solid scintillator cells
MS: TSQ 7000 instrument by Finnigan

3) GC-MS
The following equipment was used:
GC: INCOS XL instrument by Finnigan
MS: FINNIGAN MAT 8230 instrument

3) NMR-spectroscopy
The following equipment was used:
BRUKER AC 300 spectrometer, DPX 300 instrument

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 feces extracts.

Urine
For the isolation and identification of renal metabolites, individual urine samples of the male and female high dose groups were used and combined for each test. Samples were concentrated using RP-18 cartridges and purified on a glass column (XAD-4). The eluates of the RP-18 - and XAD-4-treatments were concentrated and further purified by a series of chromatograms and fractionations.

Feces
Lyophilised feces were extracted by 7 consecutive steps using approximately 150 mL of citrate buffer (pH 4) and methanol (1:1) followed by 2 consecutive steps using approximately 50 mL of the same solvents mixed in a ratio of 8:2. The extraction was carried out in a tissue homogenizer. After various steps (centrifugation, decantation, concentration, re-dissolving in methanol/water (1:1)), the solution was subjected to HPLC.

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 Mann Whitney-U-test.

Type:

absorption

Results:

The analysis of the plasma curves demonstrated that the absorption commenced immediately after oral administration. The amount of renal excretion indicated that the absorption rate was 61.3 - 66.8% of the given dose for males and 53.0 - 60.3% for females.

Type:

distribution

Results:

The maximum plasma concentrations of both sexes were reached 1 - 1.5 h after administration of the low dose and was delayed to 3 - 4 h in the the high dose group. Radioactivity was readily distributed from the plasma into peripheral compartments.

Type:

excretion

Results:

After oral administration more than 95% of the recovered radioactivity was excreted via urine and feces within 48 h in all dose groups except the high dosed females. The majority of the radioactivity was renally excreted.

Type:

metabolism

Results:

The test item was intensively metabolized. The main metabolite in urine was WAK 3583, which amounted to ca. 26% on average of the administered radioactivity in both sexes.

Details on absorption:

The plasma curves demonstrated that the absorption started immediately after oral administration. Based on the renal excretion, the absorption rate was at least 60% of the given dose for males and females. After administration of the high dose (100 mg/kg bw) the renal excretion rate decreased, which can be explained by an increased amount of unabsorbed radioactivity. For details, please refer to the attached background material (attachment 1).

Details on distribution in tissues:

Oral administration:
The maximum plasma concentrations of both sexes were reached 1 - 1.5 h after administration of the low dose, whereas after administration of the high dose this time was delayed to 3-4 h. The corresponding normalized equivalent concentrations CN varied between ca. 0.74 for the low dose groups and 0.47 in case of the high dose groups, indicating that the radioactivity was readily distributed from the plasma into peripheral compartments. This was also supported by the distribution volume under steady-state conditions (Vss), which was in most cases > 100% of the body volume. This again demonstrated that the radioactivity was quickly distributed into peripheral compartments and that the parent compound and/or its labelled metabolites had a high ability to permeate the tissues. Further the mean residence time (MRT) of the total radioactivity in the central compartment of ca. 7 to 25 h was relatively small, indicating that the redistribution into the plasma prior to elimination was also a fast process. The comparison of the plasma curves obtained after administration with the low and high doses, showed that the high dose had a significant influence on the height of the maximum as well as on the slope during the elimination phase from plasma. The lower and delayed maximum indicated a slower and possibly incomplete absorption process in the rats of the high dose group. The distribution of the dose-normalized equivalent concentrations amongst the different organs and tissues revealed differences between male and female rats in some organs or tissues, however, the concentrations in general were relatively low due to the fast elimination. The highest concentration of radioactivity was observed in kidney and liver. This distribution pattern was barely changed by dose or duration of treatment. However, the concentration in the organs and tissues of female rats treated with 100 mg/kg bw increased significantly. This is due to the fact that the elimination of the radioactivity was not finished at 48 h after administration. For details, please refer to the attached background material (attachment 2).

Intravenous administration:
The initial distribution volume (Vc) of about 7 to 10% of the total body volume was obtained from plasma curve analysis for either sex. This result indicated that the radioactivity was distributed rapidly from the plasma into peripheral compartments. For details, please refer to the attached background material (attachment 2).

Key result

Test no.:

#1

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was readily distributed to the peripheral tissues mainly liver and kidney.

Key result

Test no.:

#2

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was readily distributed to the peripheral tissues mainly liver and kidney.

Key result

Test no.:

#3

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was readily distributed to the peripheral tissues mainly liver and kidney.

Key result

Test no.:

#4

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was readily distributed to the peripheral tissues mainly liver and kidney.

Details on excretion:

Only 0.05% of the orally administered radioactivity was expired as 14C02 over a period of 48 h. Thus, the labelling position in the methylene bridge of the molecule was regarded as metabolically stable under in vivo conditions.
After oral administration more than 95% of the recovered radioactivity was excreted via urine and feces within 48 h in all dose groups except the high dosed females. The majority of the radioactivity was renally excreted (the average urine to feces ratio was 2.5:1). In general, the excretion rate was high with 78% of the radioactivity being excreted within 24 h after administration of the test substance. This can be explained by the fast distribution and redistribution processes described above. However, in the high dose groups the excretion rate decreased significantly as a result of the slower absorption process. The fecal excretion of the females of the high dose group was significantly reduced in comparison to their male counterparts. Since the renal excretion was similar in either sex and the residue in the gastrointestinal tract was higher in the female group, it was assumed that the fecal excretion in this dose group was not finalized 48 h after administration. For details, please refer to the attached background material (attachment 1).

Key result

Toxicokinetic parameters:

AUC: 5.54 h (males) and 5.82 h (females) after single oral treatment with 1 mg/kg bw

Key result

Toxicokinetic parameters:

other: Vss

Remarks:

3.76 L/kg (males) and 1.48 L/kg (females) after single oral treatment with 1 mg/kg bw

Key result

Toxicokinetic parameters:

other: MRT

Remarks:

21.7 h (males) and 9.82 h (females) after single oral treatment with 1 mg/kg bw

Key result

Toxicokinetic parameters:

other: Vc

Remarks:

0.068 L/kg (males) and 0.114 L/kg (females) after single intraveneous administration of 1 mg/kg bw

Metabolites identified:

yes

Details on metabolites:

In general, the extent of the metabolic rate decreased with dose. Thus, in the high dose groups of either sex the relative contribution of the unchanged parent compound was elevated (up to approximately 3%).

Metabolites in urine:
The main metabolite was in all dose groups but the high dosed females the glycine conjugate of 6-chloronicotinic acid (WAK 3583), which amounted to ca. 26% on average of the recovered radioactivity. Further, free 6-chloronicotinic acid occurred in significant amounts. The main metabolite found in the urine of females treated with the high dose was the glucuronide of the monohydroxylated derivative (KNO 2621). The N-hydroxylated amide compound (KNO 1893) was one of the major metabolites in the urine of all male dose groups. Although the administered radioactivity was eliminated very quickly, there was still unchanged parent compound identified in all dose groups. For details, please refer to the attached background material (attachment 4 and 5).

Metabolites in feces:
Basically, the metabolic pattern in the extracts of feces was similar to that of urine. However, not all of the renal metabolites were also found in feces. Free and conjugated 6-chloronicotinic acid were not fecally excreted. The major compounds in feces were WAK 6856 (formed by hydroxylation of the parent compound) and the unchanged parent compound (YRC 2894). For details, please refer to the attached background material (attachment 4 and 5).

Enzymatic activity measured:

not measured

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 considered valid, scientifically acceptable and appropriate for the assessment of ADME in the rat. During the study, 14C-labelled test compound was administered by single oral or intravenous doses of 1 and/or 100 mg/kg bw to male and female rats. In addition, male and female rats were treated with oral doses of 1 mg/kg bw/day (non-radioactive compound) for 14 days and 24 h after the last dose with a single oral dose of 1.0 mg/kg bw (radioactive compound). The present study demonstrated that the test compound was absorbed and readily distributed to the peripheral tissues, mainly the liver and kidney. There was no evidence of accumulation of the radioactivity in the organs or tissues. After oral administration more than 96% of the recovered radioactivity was excreted via urine and feces within 48 h in all dose groups with renal excretion being predominant. Females treated orally with 100 mg/kg bw showed a slower absorption rate and therefore an incomplete absorption of the total radioactivity 48 h after treatment. Also the remaining radioactivity in the female body was greater 1% 48 h after administration of 100 mg/kg bw of the test substance. However, the kinetic data of this dose group indicated that radioactivity was subject to further elimination from the body. The test substance was intensively metabolized, with the main metabolite being identified exclusively in the urine as glycine conjugate of 6-chloronicotinic acid (WAK 3583), which amounted to ca. 26 % on average of the administered radioactivity.

Description of key information

Rapid absorption of the test substance occurred after oral exposure along the gastrointestinal tract. This was evident in rodent studies on toxicokinetics as well as in acute and repeated-dose toxicity studies due to observed systemic effects. Following oral absorption, the test substance is readily distributed to different organs and tissues. The test compound was intensively metabolized in rodents and the elimination from the organism was fast. There is no indication of any bioaccumulation potential of the parent compound and/or its metabolites. With regard to dermal absorption, the physicochemical properties of the test substance indicated that dermal absorption is to be expected to a certain extent. This was supported by the observation of systemic effects after subacute dermal exposure. Thus, indicating that dermal absorption of the test substance is likely. Respiratory absorption is considered to be rather unlikely, 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 although the test substance has a moderate log Pow value.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

75

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 EPA Guideline 85-1, which was regarded as key study (M-000847-03-1). In this study, 4 treatment groups (in total 15 male and 5 female rats) were orally treated with the test compound, which was 14C-labelled at the positions 4 and 5 of the thiazolidine ring. In the first test group, 5 male rats received a single oral dose of 1 mg/kg bw for measuring the radioactivity in the expired air. In the second and third test group 5 male and 5 female rats were administered a single oral dose of 1 mg/kg bw, respectively. In the fourth test group, 5 male rats received a single oral dose of 100 mg/kg bw. During intervals of maximal 48 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). All biokinetic parameters were calculated for the total radioactivity measured in the plasma. The metabolites were extracted and purified from urine and feces. For quantitation and identification of metabolites, comparative high performance liquid chromatography (HPLC) with authentic reference compounds and various mass- and NMR-spectroscopic techniques were used. 

Absorption:

Based on the renal excretion of radioactivity, approximately 75% of the administered dose was absorbed after treatment with the low dose and approximately 60% after treatment with the high dose. 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.

Distribution:

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). 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.

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. 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. Therefore, the predominant route of excretion was renal. 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.

Metabolism:

16 metabolites and the parent compound were identified from urine, 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.

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, 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 from urine, whereas 5 metabolites were identified in feces. All metabolites identified in the feces were also found in the urine.

 

In a second study, the biokinetic behavior of the test substance was also investigated in rats, but the position of the used radiolabel was located on the methylene bridge (denoted methylene-14C-label). The study was conducted according to GLP and the EPA Guideline 85-1 and was therefore considered as key study (M-001080-01-1). During the study,the test substance was administered in a 0.5% tragacanth suspension at different dose levels to groups of 5 male and female rats as follows:

Group A: single oral dose of 1 mg/kg bw (for expiration test)

Group B: single intravenous dose of 1 mg/kg bw

Group C: repeated-doses of 1 mg/kg bw non-labelled compound once daily for 14 days followed by 1 mg/kg bw of a single oral radioactive dose after 24 h

Group D: single oral dose of 100 mg/kg bw

Urine, feces, expired air, blood (separated into plasma and erythrocytes), organs and tissues and residual carcass were collected. Radioactivity was measured with LSC. All biokinetic parameters were calculated for the total radioactivity measured in the plasma. The metabolites were extracted and purified from urine and feces. For quantitation and identification of metabolites, comparative high performance liquid chromatography (HPLC) with authentic reference compounds and various mass- and NMR-spectroscopic techniques were used. 

Absorption:

The plasma curves demonstrated that the absorption started immediately after oral administration. Based on the renal excretion, the absorption rate was at least 60% of the given dose. After administration of the high dose (100 mg/kg bw) the renal excretion rate decreased, which can be explained by an increased amount of unabsorbed radioactivity.

Distribution after oral administration:

The maximum plasma concentrations were reached 1 - 1.5 h after administration of the low dose, whereas after administration of the high dose this time was delayed to 3-4 h. The normalized equivalent concentrations CN varied between ca. 0.74 (low dose groups) and 0.47 (high dose groups) and the distribution volume under steady-state conditions (Vss) was in most cases > 100% of the body volume. Therefore, both values indicated that the radioactivity was readily distributed from the plasma into peripheral compartments and that the parent compound and/or its labelled metabolites had a high ability to permeate the tissues. Further the mean residence time (MRT) of the total radioactivity in the central compartment of ca. 7 to 25 h was relatively small, indicating that the redistribution into the plasma prior to elimination was also a fast process. The comparison of the plasma curves obtained after administration with the low and high doses showed that the high dose had a significant influence on the height of the maximum as well as on the slope during the elimination phase from plasma. The lower and delayed maximum indicated a slower and possibly incomplete absorption process in the rats of the high dose group. The highest concentration of radioactivity was observed in kidney and liver. The distribution pattern was barely changed by dose or duration of treatment. However, the concentration in the organs and tissues of female rats treated with 100 mg/kg bw increased significantly. This can be explained by the fact, that the elimination of the radioactivity was not finished at 48 h after administration.

Distribution after intravenous administration:

The initial distribution volume (Vc) of about 7 to 10% of the total body volume was obtained from plasma curve analysis for either sex. This result indicated that the radioactivity was distributed rapidly from the plasma into peripheral compartments.

Excretion:

Only 0.05% of the orally administered radioactivity was expired as 14C02 over a period of 48 h. Thus, the labelling position in the methylene bridge of the molecule was regarded as metabolically stable under in vivo conditions. After oral administration more than 95% of the recovered radioactivity was excreted via urine and feces within 48 h in all dose groups except the high dosed females. The majority of the radioactivity was renally excreted. In general, the excretion rate was high with 78% of the radioactivity being excreted within 24 h after administration of the test substance. This can be explained by the fast distribution and redistribution processes described above. However, in the high dose groups the excretion rate decreased significantly as a result of the slower absorption process. The fecal excretion of the females of the high dose group was significantly reduced in comparison to their male counterparts. Since the renal excretion was similar in either sex and the residue in the gastrointestinal tract was higher in the female group, it was assumed that the fecal excretion in this dose group was not finalized 48 h after administration.

Metabolism

In general, the extent of the metabolic rate decreased with dose. Thus, in the high dose groups of either sex the relative contribution of the unchanged parent compound was elevated.

Metabolites in urine:

The main metabolite was in all dose groups except for the high dosed females the glycine conjugate of 6-chloronicotinic acid (WAK 3583), which amounted to ca. 26% on average of the recovered radioactivity. Further, free 6-chloronicotinic acid occurred in significant amounts. The main metabolite found in the urine of females treated with the high dose was the glucuronide of the monohydroxylated derivative (KNO 2621). The N-hydroxylated amide compound (KNO 1893) was one of the major metabolites in the urine of all male dose groups. Although the administered radioactivity was eliminated very quickly, there was still unchanged parent compound identified in all dose groups.

Metabolites in feces:

The metabolic pattern in the extracts of feces was similar to that of urine. However, not all of the renal metabolites were also found in feces. Free and conjugated 6-chloronicotinic acid were not fecally excreted. The major compounds in feces were WAK 6856 (formed by hydroxylation of the parent compound) and the unchanged parent compound (YRC 2894).

Overall, the present study demonstrated that the test compound was absorbed and readily distributed to the peripheral tissues, mainly the liver and kidney. There was no evidence of accumulation of the radioactivity in the organs or tissues. After oral administration, more than 96% of the recovered radioactivity was excreted via urine and feces within 48 h in all dose groups with renal excretion being predominant. Females treated orally with 100 mg/kg bw showed a slower absorption rate and therefore an incomplete absorption of the total radioactivity 48 h after treatment. In addition, the remaining radioactivity in the female body was greater 1% 48 h after administration of 100 mg/kg bw of the test substance. However, the kinetic data of this dose group indicated that radioactivity was subject to further elimination from the body. The test substance was intensively metabolized, with the main metabolite being identified exclusively in the urine as glycine conjugate of 6-chloronicotinic acid (WAK 3583), which amounted to ca. 26 % on average of the administered radioactivity.

 

The results obtained in the key studies are also supported by further studies on toxicokinetics, which are shortly mentioned here for purposes of data completeness:

The distribution of the total radioactivity was determined in rats by conventional whole-body autoradiography and radioluminography (M-000660-01-1). In this GLP-compliant study, the radioactively-labelled test substance (14C-labelled in the methylene bridge) was administered to rats at single oral doses of 5 mg/kg bw and at an intravenous dose of 1 mg/kg bw. Whole-body autoradiography was performed over a period of 48 hours following oral administration and after 5 minutes following an intravenous injection. Following intravenous administration, a rapid and even distribution of the radioactivity occurred immediately (5 min) after injection. Thus, demonstrating that the parent compound possesses a high ability to permeate the tissues. This was supported by the observation that at this time the concentration in the blood was lower than in many of the organs, e.g. liver, kidney, muscle, preputial gland, adrenals, thyroid, salivary gland, and the walls of the aorta. This result indicated a fast turnover of the test compound in the rat. After oral treatment, the test compound was readily absorbed from the gastrointestinal tract and was detected in all tissues and organs. High radioactivity was observed in the entire kidney, indicated that the renal excretion had already begun.Other prominent tissues were glandular organs (adrenals, thyroid, salivary and preputial glands) and the connective tissue associated with skin, the walls of the aorta, and spinal cord. High radioactivity concentration were also detected in liver as the main metabolizing organ. Four hours after administration the radioactivity content of the stomach was still high, but also the fecal excretion commenced. The radioactivity concentration in the kidney at 4 and 8 h after treatment was consistent with the high proportion and rate of the renal excretion of the radioactivity. 24 and 48 h post application, the most prominent feature, apart from the excretory organs, was the radioactivity associated with the connective tissues in the skin, the aorta wall, and attached to the spinal cord as well as the radioactivity still present in the glandular organs like thyroid, preputial gland, and adrenals. In the quantitative part of the study, a steady decline in the equivalent concentration was observed for all organs from 1 to 48 h after treatment. The values dropped from ca. 0.7 to 12 µg/g 1 h after oral administration to < 0.16 µg/g at the end of the test period.

 

Plasma level of the test substance were also determined in a GLP-conform 15-week repeated-dose study with beagle dogs (M-003814-01-1). This study was conducted according to OECD Guideline 409. Due to marginal toxic effects observed, plasma samples were taken 13 weeks after dietary treatment with 0, 250, 1000 and 2000 ppm (1 – 4 days 4000 ppm, 5 – 14 days 0 ppm and from 15 – end of study 2000 ppm) of the test substance in order to demonstrate the efficient absorption of the test compound and the dose-dependency of the concentrations in the plasma. Samples were taken at the time points 0, 2, 4, 6 and 24 h after feeding. The peak values as measured 6 h after administration were approximately 2, 6 and 14 µg/mL for 250, 1000 and 2000 ppm respectively. Compared with the administered doses, the blood levels are regarded as very high, thus, indicating a high absorption rate.

 

For inter-species comparison an in vitro study is available, which was conducted with regards to metabolic stability and profiling in liver microsomes from rats and humans (M-003814-01-1). DIn fact, 10 µM of the test item (methylene-14C-labelled) was incubated with liver microsoms of either Wistar rats or humans using a protein concentration of 1 mg/mL. The sampling times were 0, 0.5 and 1 h 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 clearly demonstrated using a well-known biochemical reaction for a CYP3A (6(3-hydroxytestosterone that is formed from testosterone by testosterone 6(3-hydroxylase). The recovery of radioactivity was measured in both microsome incubations and amounted to > 91.3% for the 0.5 and 1 h samples. The results of the tests demonstrated that the test item was highly stable after incubation with both rat and human microsomes.Under the conditions of the test, thein vitrometabolism of the test compound was found to be comparable between rats and humans and there was no indication of the formation of a unique human metabolite. In addition, only one metabolite was detected in very low amounts of the relative percentage (< 1.7%) after incubations of the labelled test substance with both, rat and human liver microsomes. Overall, the results of this study demonstrate that phase I metabolism plays a very moderate role in the biotransformation of the test item in rat and human liver microsomes. In addition, no differences with respect to the metabolic pattern were found in both in vitro test systems.

  

Conclusion and assessment of the toxicokinetic behavior of the test substance

The assessment of the toxicokinetic behavior of the test substance was conducted to the extent that can be derived from the relevant available information inaccordance 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). This includes 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 159 mg/L at 20 °C, a molecular weight of 252.7 g/mol and a vapour pressure 3.0*10^-12 hPa at 20 °C. The octanol/water partition coefficient (log Pow) was determined around 1.4 at 24 °C, pH 7.

Absorption

In general, absorption describes the potential of a substance to diffuse across biological membranes. The most important parameters providing information on absorption are the log Pow, and water solubility. The log Pow provides information on the relative solubility of a substance in water and hydrophobic solvent (octanol), which is used as surrogate for lipids. In general, log Pow values between -1 and 4 are favorable for absorption. However, it is important to additionally consider the water solubilitywhen assessing the potential of a substance to be absorbed (ECHA, 2017).

Oral:

In general, substances with molecular weights below 500 g/mol and a log Pow between -1 and 4 are favorable for absorption provided that the substance is sufficiently water soluble. As the test substance has a molecular weight of 252.7 g/mol, a water solubility of 159 mg/L (at 20 °C) and a log Pow value of 1.4 (at 24 °C and pH 7), 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 readily 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. The stratum corneum provides the first barrier against hydrophilic compounds. In general, compounds with a log Pow value between 1 and 4 favor dermal absorption (values between 2 and 3 are optimal), particularly if water solubility is high (ECHA, 2017). Since the test substance has a moderate water solubility of 159 mg/L and a log Pow of 1.4, absorption is anticipated to be likely. However, the test substance is a solid and hindered dermal absorption has to be considered, as dry particulates first have to be dissolve into the surface moisture of the skin before uptake can begin (ECHA, 2017). Dermal toxicity data are available for acute (M-000808-01-1) and subacute toxicity (M-000824-01-1) exposure. In the acute toxicity studies, the test compound was proved to be non-toxic after dermal application to rats, which 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. However, in the subacute dermal toxicity study, the test substance caused systemic effects with regards to changes in liver and thyroid as well as transient decreased feed consumption in rodents. Although the decrease in feed consumption may also be due to stress caused by the treatment and cannot be attributed solemnly on the test substance, the observed effects indicate that dermal absorption has nevertheless occurred. Since an in vivo skin irritation study on rabbits (M-000708-03-1) revealed that the test compound is not irritating to the skin, facilitated penetration due to local skin damage can be excluded. Overall, the moderate water solubility and lipophilicity together with the observed systemic effects of the test substance after subacute dermal exposure indicate that dermal absorption of the test substance is likely.

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 3.0*10^-12 hPa at 20 °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 studies in rats is available (M-000815-01-1). This study revealed that the test substance possesses acute toxicity on uptake once via the respiratory tract in the form as dust (relatively low inhalability). This is also supported by results of a subacute inhalation toxicity study (M-241815-01-1). In this study, systemic toxicity was observed after inhalation of the test substance in form as dust. Therefore, the available data on inhalation toxicity indicate that respiratory absorption of the test substance in form of an aerosol is likely. However, due to the low vapour pressure, respiratory absorption following inhalation is not expected to be significant under normal use and handling.

Distribution

In the rat and mouse, the test substance was readily distributed over various organs. There was no evidence of accumulation. The absorption after administration of high doses (100 mg/kg bw) began slightly later than after administration of the low dose.

Excretion

The major route of elimination was renal, with 60% or more of the recovered radioactivity in the rat urine after oral administration. Fecal excretion accounted for 10 to 37% of the recovered radioactivity. Less than 1% of the administered radioactivity was found in the expired air.

As approximately up to 12.4% of the administered radioactivity remained in the organs and tissues of the rats dosed with the high doses (100 mg/kg bw) 48 h after treatment, again an incomplete absorption of total radioactivity after administration of the high dose can be assumed.

Metabolism

Although the test substance was quickly eliminated from the body, it was intensively metabolized. By using the two different labelling positions, besides the unchanged parent compound, 25 metabolites have been isolated and identified in the excreta of rats.The identification rate ranged from about 58% - 79% of the administered radioactivity using [methylene-14C]-labelled compound and about 55 - 64% of the given dose using the [thiazolidine-4,5-14C]-labelled test item.