imidacloprid (ISO); 1-(6-chloropyridin-3-ylmethyl)-N-nitroimidazolidin-2-ylidenamine

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:

06 May 1987- 11 Aug 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

other: EPA Pesticide Assessment Guidelines, Subdivision F, EPA 54019-82-025

Version / remarks:

adopted 1982

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

Radioactive labelling with 14C in the methylene moiety. For the high oral dose also 13C-labelled compound was used for better identification of metabolites.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzucht GmbH & Co KG, Borchen, Germany
- Weight at study initiation: approximately 200 g
- Housing: During the excretion studies the animals were kept in special metabolism-cages, which allowed a separate and quantitative sampling of the excreta. In all other cases animals were kept in plastic cages on wood shavings. During the nonradioactive pretreatment period the rats were housed as single animals in plastic cages.
- Diet: Altromin 1324 standard food, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: not specified
- Health status: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): at room temperature during test-period of 48 h and during the nonradio-active pretreatment period and the bile fistulation at 20 °C
- Humidity (%): 40-80
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified
- Fasting period: not specified

IN-LIFE DATES: From: 06 May 1987 To: 11 Aug 1987

Route of administration:

other: per oral, intraduodenal, intravenous

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration solution the specific amounts of labelled and unlabelled compound were dissolved in physiological saline using an ultrasonic water bath at 70 °C.
The solutions were administered per oral, intraduodenal or intravenous. The administered volume was 10 mL/kg bw for oral application and 1 mL/kg bw for intraduodenal dosage.

Duration and frequency of treatment / exposure:

Oral treatment: single low radioactive-labelled dose of 1 mg/kg bw
Oral treatment: repeated dose of non-labelled compound for 14 days followed by a single dose of the labelled compound on Day 15, all low doses of 1 mg/kg bw
Intravenous treatment: single low radioactive-labelled dose of 1 mg/kg bw
Intraduodenal treatment: single low radioactive-labelled dose of 1 mg/kg bw
Oral treatment: single high radioactive-labelled dose of 20 mg/kg bw
Oral treatment: single high radioactive-labelled dose of 20 mg/kg bw (for expired CO2 measurements)

Dose / conc.:

1 mg/kg bw/day

Remarks:

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

Dose / conc.:

20 mg/kg bw/day

Remarks:

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

No. of animals per sex per dose / concentration:

5

Control animals:

no

Positive control reference chemical:

no

Details on study design:

In addition to the experimental values, blank values were determined in all measuring procedures, which were based on blank samples prepared identically to the experimental samples.

Details on dosing and sampling:

ANALYTICAL METHOD
- Complete description: For samples of organs with weights below 500 mg or residues with a low detection limit, samples were weighed and combusted in an oxygen atmosphere using an oxidizer. Radioactivity in trapped combustion gases was measured by liquid scintillation counter (LSC). Fatty organs and tissues were solubilized by means of a tissue solubilizer. Radioactivity from aliquots was measured by LSC. Liquid samples were added with scintillation gel and measured by LSC.

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, feces, bile, plasma, 14CO-expired in ethanolamine, blood (separated into plasma and erythocytes), spleen, gastrointestinal tract, organs (liver, kidney, testis, ovaries, uterus, muscle, bone, heart, lung, brain, skin, redidual carcass, renal fat)
- Time and frequency of sampling:
Plasma samples were taken at 5, 10, 20, 40 min and 1, 1.5, 2, 3, 4, 6, 8, 24, 32 and 48 h post application.
Urine was sampled in intervals of 0 – 4, 4 – 8, 8 – 24, 24 – 32 and 32 - 48 h.
Faeces was sampled in periods of 0 - 24 and 24 –48 h after dosage.
- From how many animals: all animals, samples were not pooled
- Method type(s) for identification: Measurement of solid samples using LSC and liquid samples were added with scintillation gel and measured by LSC
- Limits of detection and quantification: not specified

Statistics:

In all cases the non-parametrical LJ-Test (Mann & Whitney) was used.
Furthermore, please refer to "Any other information on materials and methods incl. tables".

Type:

absorption

Results:

Radioactivity was absorbed almost completely (ca. 95 % of the orally administered dose) by male and female rats. The time course of absorption was described by a half-life of ca. 35 minutes.

Type:

distribution

Results:

The maximum concentration of the radioactivity in the plasma was reached between 1.1 and 2.5 hours. Radioactivity was further distributed rapidly to the organs/tissues, mainly in liver, kidneys, lungs and the skin.

Type:

excretion

Results:

The excretion is fast and mainly renal. More than 90 % of the renal radioactivity were excreted within 24 h after dosage. The total excretion after 48 h amounted to ca. 96 % of the given dose, approximately 75 % with the urine and 21 % with the feces.

Details on absorption:

After oral administration of both, the high and low dose of radioactive-labelled compound, a maximum dose normalized concentration of radioactivity in the plasma was reached between 1.1 and 2.5 hours. In all cases the peak concentration was low with an average of 0.73 mg/L, compared to the equidistribution of 1. From the experiment using bile-fistulated rats and intraduodenal administration the amount of absorbed radioactivity was calculated to be 95 % of the given dose. This is in good agreement with the estimations for the
oral and intravenous tests. In all dose groups under investigation the rate of absorption can be described with an average half-life of approximately 35 minutes taking into account a lagtime of less than 2.5 minutes. For details, please refer to the attached background material.

Details on distribution in tissues:

The radioactivity of the test compound was rapidly distributed from the intravascular space to the peripheral tissues. After intravenous injection of 1 mg/kg bw, an apparent initial distribution volume (Vc) of about 84 % of the total body volume was obtained from plasma curve analysis for either sex. This result indicated that the radioactivity was readily distributed from the plasma into peripheral compartments.
The distribution volume under steady-state conditions (Vss) was roughly in the same order of magnitude as the apparent initial distribution volume (Vc) after intravenous administration with the exception of the male rats, which received a single oral dose of 1 mg/kg bw. This supports the assumption that the radioactivity was distributed very quickly into peripheral compartments. It also means that the parent compound and/or its labelled metabolites have a high ability to permeate the tissues. However, the small mean residence time (MRT) of the total radioactivity in the central compartment (plasma) which varied between about 9 and 17 hours indicated that the redistribution into the plasma prior to elimination, mainly via the kidney, was also a fast process.
The remaining radioactivity in the body excluding the gastrointestinal tract at sacrifice 48 hours after oral and intravenous administration was in all dose groups below 1 % of the recovered radioactivity. However, from the kinetics of the renal excretion and of the elimination behaviour of the total radioactivity from the plasma it can be concluded, that also the remaining radioactivity in the body was subject to further elimination. At the end of the test period (48 h post application) the average dose-normalized concentration in the body excluding gastrointestinal tract was about 0.005 mg/L independent of the route of administration. Most of the investigated organs and tissues showed lower values. For details, please refer to the attached background material.

Key result

Test no.:

#1

Transfer type:

other: transfer observed from plasma into organs

Observation:

other: Radioactivity was distributed rapidly to the peripheral tissues mainly liver, kidney, lung and skin.

Details on excretion:

In all tests of this study the elimination of the total radioactivity from the plasma could be approximated by a combination of two exponential terms, from which elimination half-lives were calculated. These half-lives varied between ca. 2.6 to 3.6 and 26 to 118 hours, respectively. The radioactivity was readily eliminated from the body.

After intravenous administration about 91.4 % of the recovered radioactivity was excreted via urine and faeces within 48 hours in the 1 mg/kg bw bw dose group. The majority of the radioactivity was renally excreted [average ratio: 4 : 1 (urine : faeces)].

Within 48 h after oral administration about 96 % of the given dose was excreted via urine and faeces. There were no differences between female and male rats. More than 90 % of the renal radioactivity was already excreted during 24 hours after dosage. The reason for this behaviour is the fast distribution and redistribution and the good water solubility of the parent compound and its metabolites. The residual radioactivity in the body excluding the gastrointestinal tract at sacrifice was about 0.5 % and in the gastrointestinal tract about 0.06 % of the given dose on average.

Bile-fistulated rats excreted only 4.7 % of the administered dose with the faeces, 56.4 % via the urine and ca. 36 % with the bile. The biliary excretion was very rapid. More than 90 % of the biliary radioactivity was already excreted after 12 hours. The course of elimination can be described by two exponential terms with half-lives of 2.9 and 10.1 hours, respectively. The difference of the renally excreted radioactivity between bile-cannulated and ‘intact’ animals (57.5 versus 77.8 % of the recovered amount) is a strong hint to the existence of an enterohepatic circulation of the radioactivity. During this circulation a part of the biliary radioactivity is being re-absorbed from the gastrointestinal tract and the major part thereof then being eliminated via the kidney.
The investigation of the expired air (CO2) over a period of 48 hours did not reveal significant amounts of radioactivity. This demonstrates that the chosen labelling position within the molecule was stable with respect to the formation of volatile C-1-fragments. For details, please refer to the attached background material.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 1 mg/kg bw i.V.: 5.517 h (males) and 6.117 h (females)

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 1 mg/kg bw i.v.: 2.70 h (males) and 3.23 h (females)

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 1 mg/kg bw i.v.: 60.18 h (males) and 28.58 h (females)

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 1 mg/kg bw p.o.: 5.472 h (males) and 5.771 h (females), 20 mg/kg bw p.o.: 4.977 h (males) and 6.503 h (females)

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 1 mg/kg bw p.o.: 2.59 h (males) and 3.34 h (females), 20 mg/kg bw p.o.: 3.05 h (males) and 3.59 h (females)

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 2nd: 1 mg/kg bw p.o.: 118.09 h (males) and 39.75 h (females), 20 mg/kg bw p.o.: 31.35 h (males) and 72.57 h (females)

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 1 mg/kg bw p.o.: 1.46 h (males) and 1.11 h (females), 20 mg/kg bw p.o.: 1.59 h (males) and 1.66 h (females)

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 1 mg/kg bw p.o. with pretreatment: 5.750 h (males) and 5.944 h (females)

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 1 mg/kg bw p.o. with pretreatment: 3.26 h (males) and 3.40 h (females)

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 2nd: 1 mg/kg bw p.o. with pretreatment: 25.84 h (males) and 43.54 h (females)

Key result

Test no.:

#3

Toxicokinetic parameters:

Tmax: 1 mg/kg bw p.o. with pretreatment: 2.43 h (males) and 2.05 h (females)

Metabolites identified:

not measured

Details on metabolites:

not applicable

Enzymatic activity measured:

not measured

Conclusions:

The biokinetic behaviour of the test compound was investigated in a GLP-compliant study on rats according to EPA Pesticide Assessment Guidelines (Subdivision F, EPA 54019-82-025, adopted 1982). The study is therefore 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, lung and skin. In addition, excretion is also fast, occurring mainly via the urine and to a lesser extend via feces.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Jan 1990 - 11 Jan 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

adopted 1982

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

[imidazolidine-4,5-14C] labelled compound

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Commonly used for toxicological studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzucht GmbH & Co KG, Borchen, Germany
- Weight at study initiation: approximately 200 g
- Housing: Metabolism cages, which allow for the quantitative separation of urine and feces.
- Diet: Altromin 1324 standard food (Fa. Altrogge, Lage, Germany), 15 g per day
- Water: tap water, ad libitum
- Acclimation period: not reported
- Health status: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported
- Fasting period: not reported

Route of administration:

oral: gavage

Vehicle:

other: physiological saline (1.0 mg/kg bw) or 0.5 % Tragacanth suspension (150 mg/kg bw)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amounts of the substance were either dissolved in physiological saline using an ultrasonic water bath at 70°C or suspended homogeneously in a 0.5 % Tragacanth suspension. For the 150 mg/kg bw solution, the labelled substance was weighed in to reach a concentration of 0.1 mg/mL and the unlabelled substance had a concentration of 14.9 mg/mL, resulting in a total concentration of 15 mg/mL for the substance.
The administered volume was 10 mg/kg bw.

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

male rats, collection of CO2, urine, feces and carcass

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

male rats; collection of urine, feces and organs; time-dependent investigation of the radioactivity concentration in the plasma

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

female rats; collection of urine, feces and organs; time-dependent investigation of the radioactivity concentration in the plasma

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

male rats; collection of urine, feces and organs; time-dependent investigation of the radioactivity concentration in the plasma

No. of animals per sex per dose / concentration:

5

Control animals:

no

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: Not reported

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, feces, expired air, blood, plasma, erythrocytes, carcass
- Time and frequency of sampling: urine and feces: 0-24 h and 24-48 h,

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: for structure elucidation, urine collected from 0 to 24 h was analyzed
- From how many animals: five pooled animals of the high dose group (150 mg/kg bw)
- Method type(s) for identification HPLC (HP 1090 Liquid chromatograph and HP 1050 Liquid chromatograph, MS (in line with HPLC, using the electron impact method, 1H-NMR (Bruker AM 300 NMR-spectrometer).
- Limits of detection and quantification: not reported

ANALYTICAL METHOD
MEASUREMENT OF LIQUID SAMPLES USING LIQUID SCINTILLATORS
As liquid scintillator counters, Beckman LS 7800, Philips PW 4700 and LKB Rack Beta 1219 Spectral were used, the scintillator volume was 7 mL, urine was used in volumes of 0.1 - 0.5 mL, plasma at 0.5 mL, and for plasma microsamples, ca 30 mg were analyzed.

MEASUREMENT OF SOLID SAMPLES USING LIQUID SCINTILLATORS
- combustion: for samples below 500 mg or for an analysis of residues with a low detection limit, done with Oxidizer 306 (for liver, kidney, testes, muscle, heart, brain, skin, carcass, faeces collected up to 24 hours post administration) or with OX 300 (for erythrocytes, spleen, gastrointestinal tract, bone, lung, faeces collected later than 24 h post application)

Fatty organs or tissues (renal fat) were solubilized by means of a tissue solubilizer (e.g. BTS 450R, Beckman).

As liquid scintillator counters, Beckman LS 7800, Philips PW 4700 and LKB Rack Beta 1219 Spectral were used.

- the limit of detection was determined by measuring blank samples collected before administration of the test substance.
- relative concentration was analyzed by dividing the radioactivity measured/grams of plasma or tissue by the radioactivity administered/grams of body weight
- equivalent concentration is calculated by multiplying the relative concentration with the dose in mg/kg bw.

Preliminary studies:

no

Type:

absorption

Results:

The test item was rapidly absorbed. Peak plasma concentrations were detected after 1/1.5 h in males/females for the low dose and after 4 h in males for the high dose (single exposure).

Type:

distribution

Results:

The highest radioactivity was found in the organs and tissues responsible for metabolism and excretion: liver, gastrointestinal tract and kidney.

Type:

metabolism

Results:

The item was metabolised, the parent compound and 4 metabolites were identified in urine. The main compound found in urine was a metabolite, followed by the parent compound.

Type:

excretion

Results:

Excretion was fast and almost complete at 24 h post dosing. Ca. 97-99% of the administered radioactivity were excreted with urine and feces. The main excretion route was urinary with 88 - 94% of the administered radioactivity measured 48 h after dosing.

Details on absorption:

Recovery
For all treatments, recovery was between 99.32 and 100.72%. Most of the radioactivity was found in the urine (88.2-93.79%) and 6.3-11.24% were found in feces. Up to 0.12% was found in the gastrointestinal tract and 0.59-1.02% remained in the carcass (exclusive the GIT).

Summarized results can be found in Attachment 1.

Absorption
The test item was rapidly and nearly quantitatively absorbed from the gastrointestinal tract of male
and female rats in all dose groups. Absorption started immediately after oral administration as shown by the time course of the relative plasma concentrations of radioactivity (see Attachment 2). A maximum peak of plasma concentration was observed after 1 h for males 1.5 h for females of the low dose group and after 4 h for the high dose group. While the maximum dose-normalized concentration values are comparable between males and females (P = 0.94 and 0.89, respectively) treated with low dose, it is significantly lower in the plasma of male rats (P = 0.39) after administration of the high dose (see Attachment 3). Moreover, the plasma-concentration curve of high dose animals is broadened compared to the low dose animals. It is suggested that the higher dose delayed absorption.

Details on distribution in tissues:

Elimination from plasma differed in low and high dose rats. The terminal elimination half-lives of the low dosed rats varied between 21.34 h (females) and 24.89 h (males), while the half-life of radioactivity in high dose rats was 9.04 h. As already mentioned above, for high dosed animals the plasma maximum is delayed and broadened and the Mean Residence Time (MRT) in the central compartment is higher (14.25 h) as compared to the low dosed rats (average of 8.8 h). Other plasmakinetics are comparable and renal clearance is fast for all conditions (Attachment 3).

At the end of the observation period (after 48 h), only small amounts of the 14C-labelling are found in the organs of the rat, 1% or below of the given dosis. The highest amounts were found in liver (up to 0.04%), kidney (up to 0.02%), lung (up to 0.02%) and skin (up to 0.02%) and - in case of males - fat tissues (0.01%). Generally, females showed lower 14C-residues than males.

Summarized results can be found in Attachment 4.

Key result

Test no.:

#1

Transfer type:

other: transfer was observed from blood into organs

Observation:

other: small amounts of radioactivity were found in liver, kidney, lung and skin

Details on excretion:

Radioactivity was mainly excreted via the urine. In the low dose group males and females excreted 88.8 and 92.6% (respectively) of the administered dose after 24 h, with 76.3 (males) and 70.1% excretion after 8 h. After 48 h, low dose animals had excreted 89.88% (males) and 93.79% (females) of the dose via the urine.
In the high dose group, 75.76% of the administered dose were excreted via the urine in the first 24 h and 90.69% after 48 h.
Excretion via feces was minorly important but also happened mainly in the first 24 h (8.04% in males of low dose group, 6.08% in females of low dose group, 4.4% males high dose group vs 8.44% males low dose, 6.3% females low dose and 7.5% males high dose after 48 h).
For an additional group of males receiving 1 mg/kg bw, expired air was trapped to analyze excretion via exhalation. There, 0.111% of the administered dose was recovered.

Summarized results can be found in Attachment 1.

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 5.5-7.1 h

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 21.34-24.89 h (low dose animals)

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 9.04 h (high dose animals)

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 1 h (males low dose), 1.5 h (females low dose), 4 h (males high dose)

Metabolites identified:

yes

Details on metabolites:

Results for female and male low dose animals are very similar. In combined urine samples of the high dose group, the parent compound was detected with 14.2% of the total radioactivity in the urine. Two main compounds were found that were analyzed by NMR and mass spectrometry.
In both KNO 0523 (30% of renal radioactivity in low dose animals, 20% in high dose males) and KNO 0524 (18.4% of renal radioactivity), the aromatic moiety got lost. KNO 0524 was identical with one reference compount while KNO 0523 differed from KNO 0524 by a double bond in the imidazole ring.
Apart from these two molecules, the parent compound and two more metabolites were detected in the HPLC analysis of the native urine. These metabolites were already identified in the previously performed study M-024189-01-1 and named WAK 4103 and NTN 35884 and they made up 14.3 and 8.4% of renal radioactivity, respectively.
The remaining radioactivity is mainly from one metabolite which could not be identified.
From the results obtained, a biotransformation scheme could be established and is shown in the attached background material (Attachment 6).

Metabolisation in the feces was not assessed since, as described above, excretion via feces was minor compared to renal excretion. Moreover, study M-024189-01-1 already identified main metabolites in feces.

This study was performed as an additional study to M-024189-01-1, where the test substance was labelled at the methylene with 14C. There, the majority of the radioactivity was absorbed and subsequently eliminated with the bile into the lumen of the intestinal tract. Since the excretion pattern was similar for both studies, it can be suggested that also in the present case the fecal radioactivity is mainly of biliary origin. Also in terms of pharmacokinetics no major differences were found between the two studies. AUC was in the same range after approximately 6 h, which means that also the parameters derived from this value were comparable. In contrast, excretion via urine was found to be higher in present study as compared to the one using methylene labelled test substance (ca. 90% vs ca. 75%). Even though radioactivity in organs after sacrifice was still low, it was higher after administration of the [imidazolidine-4,5-14C] labelled compound compared to the methylene labelled test substance. But the kinetics of renal secretion suggest that this moiety is also eliminated via the urine. 

 

Summarized data can be found in Attachment 5. 

Conclusions:

The biokinetic studies in rats showed that imidacloprid is rapidly and almost completely absorbed from the intestinal lumen. Also the elimination from the organism is fast and complete, with no indication of any bioaccumulation potential of the parent compound and/or its metabolites. Ca 90% of the administered radioactivity is excreted with the urine, the remainder was recovered in the feces. Most of the fecal radioactivity was expected to origin from biliary excretion. The main amount of radioactivity was excreted in the first 24 h after administration.

Peak plasma concentrations were reached within 1/1.5 to 4 hours after administration for low and high dose animals, respectively. The radioactivity was rapidly distributed from the intravascular space to the peripheral tissues and organs. At the end of the test period of 48 h radioactivity concentrations in the tissues were very low. Levels above average were only observed in the contents of the gastrointestinal tract, liver, kidney, skin and lung. The extent of the permeation through the blood-brain barrier was only very limited.

Four metabolites and the parent compound were identified. The metabolite KNO 0523 was the main molecule found with 20-30% of the detected radioactivity in the urine, followed by the parent compound. The remaining metabolites compromised 8.6-14.2% each. The remaining readioactivity in the urine was contributed to a non-determinable metabolite. However, from the results obtained in this study and the results obtained in from a further study (please refer to study M-024189-01-1), a scheme of metabolisation could be proposed. Pharmacokinetic parameters could be determined and were similar to the ones found in the previously performed study.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Apr 1987 - 05 Sep 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

metabolism

Qualifier:

according to guideline

Guideline:

other: EPA OPP 85-1

Version / remarks:

adopted 1982

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

Radioactive labelling with 14C in the methylene moiety. For the high oral dose also 13C-labelled compound was used for better identification of metabolites.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann Versuchstierzucht GmbH & Co KG, Borchen, Germany
- Weight at study initiation: approximately 200 g
- Housing: During the excretion studies the animals were kept in special metabolism-cages, which allowed a separate and quantitative sampling of the excreta.
- Diet: Altromin 1324 standard food, ad libitum
- Water: tap water, ad libitum
- Acclimation period: not specified
- Health status: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): at room temperature during test-period of 48 h and during the non-radioactive pretreatment period and the bile fistulation at 20 °C
- Humidity (%): 40-80
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified
- Fasting period: not specified

IN-LIFE DATES: From: 06 May 1987 To: 11 Aug 1987

The samples were derived from a previous study (M-024189-01-1).

Route of administration:

other: oral and intravenous

Vehicle:

physiological saline

Duration and frequency of treatment / exposure:

Oral treatment: single low radioactive-labelled dose of 1 mg/kg bw
Oral treatment: repeated dose of non-labelled compound for 14 days followed by a single dose of the labelled compound on Day 15, all low doses of 1 mg/kg bw
Intravenous treatment: single low radioactive-labelled dose of 1 mg/kg bw
Oral treatment: single high radioactive-labelled dose of 20 mg/kg bw

Dose / conc.:

1 mg/kg bw/day

Remarks:

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

Dose / conc.:

20 mg/kg bw/day

Remarks:

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

No. of animals per sex per dose / concentration:

5

Control animals:

no

Positive control reference chemical:

No

Details on study design:

In addition to the experimental values blank values were determined in all measuring procedures, which were based on blank samples prepared identically to the experimental samples.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, feces
- Time and frequency of sampling: Urine was collected separately for each animal under cooling in the intervals 0 - 4, 4 - 8, 8 - 24, 24 - 48 h p.a. (post application); the rinsing solutions were collected in the urine containers.
For structural elucidation, urine was pooled from 0-6 h and from 0-24 h.
Feces were collected for each animal from 0-24 h and 24-48 h (lyophilized and homogenized before extraction)
- From how many animals: Urine metabolism elucidation pooled, feces pooled for each dose group and sex
- Method types for identification: HPLC (HP 1090 liquid chromatograph) in line with a radioactivity flow-through detector Ramona 4 or 6. If needed, urine samples were purified via adsorption chromatography on an "Extrelut" column. Identification was carried out by mass spectrometry (MS, Finnigan-MAT 8230 mass spectrometer), sometimes combined with gas chromatography (Varian 3700 gas chromatograph) or using NMR (Bruker NMR spectrometer AM 360).
- radioactivity measurements: scintillation measurement (LS-counting), solid samples were combusted
- Limits of detection and quantification: not reported

Skin, main organs/tissues, carcass and gastrointestinal tract were assayed for radioactivity after lyophilization and combustion of aliquots followed by liquid scintillation counting of the trapped 14CO2. This data was used for balancing purposes.

Statistics:

No data provided

Type:

metabolism

Results:

The parent compound as well as five major and five minor metabolites were identified in urine and feces.

Details on absorption:

Not assessed

Details on distribution in tissues:

Not assessed

Details on excretion:

The excretion was assessed in study M-024189-01-1. Briefly, it was found, that 90-98% of the administered radioactivity was excreted within 48 h post administration, less than 1% of the radioactivity remained in the carcass. More than 90% of radioactivity was recovered in the first 24 h, so that urine and feces for this interval were used for metabolic identification.

Summarized data can be found in Attachment 1.

Metabolites identified:

yes

Details on metabolites:

The parent compound, 5 major and 5 minor metabolites were identified in the urine and feces. The major metabolites were 4- and 5-OH (WAK 4103)-metabolized test substance, an olefinic metabolite (NTN 35884), 6-chloronicotinic acid, and glycine conjugate of 6-Chloronicotinic acid (WAK 3583). Metabolites with a code were also available as reference materials.

Minor metabolites were glycine conjugate of 6-methylmercaptonicotinic acid, mercapturic acid derivative of 6-chloronicotinic acid, 6-methylmercaptonicotinic acid, 6-hydroxynicotinic acid and cyclic urea (NTN 33519).

In fact, in urine, all the metabolites mentioned above were found whereas in the feces, 6-chloronicotic acid and its glycine conjugate could not be identified.

Quantification results
Identification rates ranged from circa 74% of the recovered radioactivity in the pretreated female rats to 82 % in the intravenously low dosed male animals. When rats were administered the dose intravenously, the amount of the parent compound in excreta was higher as compared to the oral route (probably due to the absence of the first-pass effect). With respect to recovered but non-identifiable radioactivity, the rate ranged between 10 and 14.5%, so that the overall recovery of radioactivity among all treated groups was between 87 and 92% of the initial amount.
6-Chloronicotinic acid and its glycine conjugate (WAK 3583) were detected in higher amounts in males than in females at the lower dose. When rats were pretreated with the non-labelled compound, decreased glycine conjugation was observed, indicating a saturation effect.
In orally administered high dose females, less metabolism of the test substance was observed compared to males. Females of this dose group excreted more of the parent compound while the olefinic metabolite was found at higher concentrations in the excreta of high dosed males.
30% of the total excreted radioactivity via urine were 6-Chloronicotinic acid and its glycine conjugate.

Summarized data can be found in Attachment 2.

Based of the results, a biotranformation pathway has been proposed by the authors of the study, which can be found in Attachment 3.

Conclusions:

This study was performed to identify the metabolite profile of the test substance in male and female rats treated either orally or intravenously. Samples were derived from a study previously conducted that assessed absorption, distribution and excretion of the substance (M-024189-01-1).

As already shown (M-024189-01-1), most of the radioactivity administered was excreted, with only 1% remaining in the carcass after 48 h. 14% of the parent compound were excreted, suggesting rapid passage of the test substance through the body. This is also supported by the fact that 90% of radioactivity was recovered after 24 h. Main excretion was via urine as demonstrated by the previous study, and to a lesser extend, also via feces.

Of the urinary and fecally excreted rate of radioactivity, about 74 to 82% could be identified, consisting of the parent compound, 5 identified major metabolites and 5 identified minor metabolites. The major identified metabolites comprised 5-OH-Imidacloprid, 4-OH-Imdiacloprid, an olefinic metabolite, 6-Chloronicotinic acid and its glycine conjugated metabolite. The minor identified metabolites comprised a glycine conjugate of 6-Methylmercaptonicotinic acid, a mercapturic acid derivative of 6-Chloronicotinic acid, a 6-methylmercaptonicotinic acid, 6-hydroxynicotinic acid and a cyclic urea metabolite.

The metabolites 6-Chloronicotinic acid and its glycine conjugate make up ca. 30% of the recovered radioactivity in the urine. These resulted from the first oxidative cleavage of the parent compound and were not found in feces. Of the minor metabolites, 6-hydroxynicotinic acid and its mercapturic acid derivative were probably derived from a glutathione conjugate. 6-Methylmercaptonicotinic acid and its glycine conjugate were also found in urine and feces, the latter being more important quantitatively (5.6%).
The test substance can also be metabolized by hydroxylation of the imidazole ring (4- or 5-position), but no dihydroxylated metabolite was found. The "olefinic" metabolite NTN 35884 is yielded by elimination of H2O and excreted via urine and feces.

Little sex differences were found. In the orally-treated high dose, males showed a higher rate of metabolization than females. The parent compound was found in lower amounts and the formation of the metabolite NTN 35884 was increased in males compared to females. All identified metabolites were found in each dose group and both sexes.

A biotransformation pathway could be postulated. The study was conducted according to EPA Pesticide Assessment Guidelines, Subdivision F and under GLP.

Description of key information

Absorption of the test substance is to be taken into consideration after oral and inhalation exposure, whereas dermal absorption is 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. With respect to the remaining routes, 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 the rat is very high 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

Absorption rate - dermal (%):

10

Additional information

Study summaries

The toxicokinetic and metabolic behavior of the test substance was investigated in the rat in various GLP-compliant studies according to the US-EPA Pesticide Assessment Guidelines, the OECD Guideline and the Guideline of the Japanese registration. These studies are summarized below.

Of these studies, the following three studies addressing absorption, distribution, metabolism and excretion were retained as key studies.

In the first key study, absorption, distribution, and excretion of the test substance in rats were investigated. Therefore, groups of 5 rats per sex were treated with single oral doses of 1 or 20 mg/kg bw and an intravenous dose of 1 mg/kg bw of the radioactive-labelled test substance in the methylene-moiety (M-024189-01-1). In order to quantitatively determine the absorbed amount of the total radioactivity and to measure the rate and extent of biliary excretion, one test group of 5 male rats was bile-fistulated and subsequently intraduodenally administered with a single dose of 1 mg/kg bw of the labelled test compound. A further group of 5 male rats received a single oral dose of 20 mg/kg bw of the radioactive-labelled compound to determine the radioactivity in the expired air. In an additional test, groups of 5 rats per sex were treated with 14 daily oral doses of 1 mg/kg bw of the unlabelled compound followed by a single oral dose of labelled material at the same level, on Day 15.

Absorption:

After oral administration of both, the high and low dose of labelled test substance the maximum dose normalized concentration of radioactivity in the plasma was reached between 1.1 and 2.5 hours. In all cases, the peak concentration was low with an average of 0.73 mg/L, compared to the equidistribution of 1. From the experiment using bile-fistulated rats and intraduodenal administration the amount of absorbed radioactivity was calculated to be 95 % of the given dose (for DNEL calculation, complete absorption was considered). This is in good agreement with the estimations for the oral and intravenous tests. In all dose groups under investigation the rate of absorption can be described with an average half-life of approximately 35 minutes taking into account a lagtime of less than 2.5 minutes. The high absorption of the test item is further confirmed by the fact that the majority of the administered radioactivity was again renally excreted and thus, recovered in urine.

Distribution:

The radioactivity of the labelled test substance was rapidly distributed from the intravascular space to the peripheral tissues. After intravenous injection of 1 mg/kg bw, an apparent initial distribution volume (Vc) of about 84 % of the total body volume was obtained from plasma curve analysis for either sex. This result indicated that the radioactivity was readily distributed from the plasma into peripheral compartments. The distribution volume under steady-state conditions (Vss) was roughly in the same order of magnitude as the apparent initial distribution volume (Vc) after intravenous administration with the exception of the male rats, which received a single oral dose of 1 mg/kg bw. This supports the assumption that the radioactivity was distributed very quickly into peripheral compartments. It also means that the parent compound and/or its labelled metabolites have a high ability to permeate the tissues. However, the small mean residence time (MRT) of the total radioactivity in the central compartment (plasma) which varied between about 9 and 17 hours indicated that the redistribution into the plasma prior to elimination, mainly via the kidney, was also a fast process. The remaining radioactivity in the body excluding the gastrointestinal tract at sacrifice 48 hours after oral and intravenous administration was in all dose groups below 1 % of the recovered radioactivity. However, from the kinetics of the renal excretion and of the elimination behavior of the total radioactivity from the plasma it can be concluded, that also the remaining radioactivity in the body was subject to further elimination. At the end of the test period (48 hours post application), the average dose-normalized concentration in the body excluding gastrointestinal tract was about 0.005 mg/L independent of the route of administration. Most of the investigated organs and tissues showed lower values. The highest value was found in the kidney and the lowest value was detected in the brain. Identical patterns of distribution of total radioactivity were found in organs and tissues sampled at different times (40 minutes - 6 hours) following a single oral administration of 20 mg/kg bw. In this test all organs reached maximum concentrations already 40 minutes after application. In summary it was found that the radioactivity administered with the test substance was very rapidly absorbed from the intestinal lumen and also readily distributed from the plasma to organs and tissues.

Excretion:

In all tests of this study the elimination of the total radioactivity from the plasma could be approximated by a combination of two exponential terms, from which elimination half-lives were calculated. These half-lives varied between ca. 2.6 to 3.6 and 26 to 118 hours, respectively. The radioactivity was readily eliminated from the body. After intravenous administration about 92 % of the recovered radioactivity was excreted via urine and feces within 48 hours in the 1 mg/kg bw dose group. The majority of the radioactivity was renally excreted [average ratio: 4 : 1 (urine : feces)]. Within 48 hours after oral administration about 96 % of the given dose was excreted via urine and feces. There were no differences between female and male rats. More than 90 % of the renal radioactivity was already excreted during 24 hours after dosage. The reason for this behavior is the fast distribution and redistribution and the good water solubility of the parent compound and its metabolites. The residual radioactivity in the body excluding the gastrointestinal tract at sacrifice was about 0.5 % and in the gastrointestinal tract about 0.06 % of the given dose on average. Bile-fistulated rats excreted only 4.7 % of the administered dose with the feces, 56.4 % via the urine and ca. 36 % with the bile. The biliary excretion was very rapid. More than 90 % of the biliary radioactivity was already excreted after 12 hours. The course of elimination can be described by two exponential terms with half-lives of 2.9 and 10.1 hours, respectively. The difference of the renally excreted radioactivity between bile-cannulated and ‘intact’ animals (57.5 versus 77.8 % of the recovered amount) is a strong hint to the existence of an enterohepatic circulation of the radioactivity. During this circulation a part of the biliary radioactivity is being re-absorbed from the gastrointestinal tract and the major part thereof then being eliminated via the kidney. The investigation of the expired air (CO2) over a period of 48 hours did not reveal significant amounts of radioactivity. This demonstrates that the chosen labelling position within the molecule was stable with respect to the formation of volatile C-1-fragments.

 

The aim of the second key study (M-024182-01-1) was to identify the metabolite profile of the test substance in male and female rats. In fact, the samples used therefore were taken from a study previously conducted that assessed absorption, distribution and excretion of the substance (please refer to the first key study M-024189-01-1). The dose groups considering were the following:

• single intravenous low dose of 1 mg/kg bw;
• single oral low dose of 1 mg/kg bw;
• 14 daily single oral low non-radioactive doses, followed by a radioactive dose on the 15th day;
• single oral high dose of 20 mg/kg bw.

Each group consisted of 5 male and 5 female animals. The metabolism of labelled test compound in the rat was investigated in the excreta of the animals. Besides the unchanged parent compound, different metabolites were identified. Also, the quantitative distribution as percent of the recovered radioactivity of the test substance and the identified metabolites in the excreta (sum of urine and feces) in rats 24 hours after administration were determined. After administration of the low doses to rats the metabolism of the test substance showed only very little sex-related differences. In contrast, some sex-related differences were noticed in the high dose group, regarding both, the excretion pattern - females showed a somewhat higher renal elimination rate than males - and the metabolic profile. Male animals showed a higher tendency to metabolize the test compound, thus the amount of parent compound was significantly lower as compared to females and as a consequence, the formation of the olefin metabolite was increased. The formation of the other biotransformation products was not affected by the high dose in either sex. The average identification rate of all dose groups in both, urine and feces was approximately 78 % of the recovered radioactivity. All identified metabolites were found in each dose group and both sexes. Thus, the differences found were quantitative rather than qualitative in nature. The main metabolites include 6-chloronicotinic acid (M14) and its glycine conjugate (M15) which were only found in urine. The monohydroxylated metabolites, -5-hydroxy and -4-hydroxy were detected at similar concentrations as the unchanged parent compound. All other biotransformation products were quantitatively of minor significance.

 

In the third key study, the biokinetic behavior of the test substance was also investigated on rats (M-024167-01-1). The compound was differentially labelled compared to the first key study with 14C in the 4- and 5-position of the imidazolidine moiety. This substance was orally administered to male and female rats at a dose level of 1 mg/kg bw and additionally to male rats at a dose level of 150 mg/kg bw.

Absorption:

The absorption based on the renal elimination was estimated to be more than 90 % of the given dose. Taking the results of the study using the methylene-14C label into consideration, a complete absorption of the administered radioactivity from the lumen of the gastrointestinal tract can be assumed. After oral administration of the labelled test substance, the maximum plasma concentration was reached between 1 hour (male rats, low dose), 1.5 hours (female rats, low dose) and 4 hours (male rats, high dose). While the maximum dose normalized concentrations are comparable between males (0.94) and females (0.89) treated with low dose, they are significantly lower in the plasma of male rats (0.39) after administration of the high dose. This is probably due to an incomplete and delayed absorption of the radioactivity from the lumen of the gastrointestinal tract after oral dosage of the exaggerated dose of 150 mg/kg bw.

Distribution:

The elimination of the total radioactivity from the plasma into peripheral compartments depended on the different dose levels. The radioactivity was quickly eliminated from the plasma with terminal half-lives between 9 hours (high dose) and 25 hours (low dose). The mean residence time (MRT) in the central compartment (plasma) is 14.3 hours for the high dose rats and 8.8 hours for the low dose rats. The shorter half-life and the longer MRT in the high dose rats are an indication for the delayed and incomplete absorption. The other pharmacokinetic parameters do not differ in a significant way between both dose groups. The total and the renal clearance demonstrate the fast elimination of the total radioactivity from the plasma and hence from the body. The 14C-labelled residues in the body at the end of the investigation period 48 hours after oral administration were at or below 1 % of the given dose. However, from the kinetics of the renal excretion it can be concluded, that also these amounts of radioactivity will be subjected to further excretion. The average dose-normalized concentration in the plasma was calculated as 0.006 at sacrifice. Most of the investigated organs and tissues showed lower values. Similar or higher concentrations were measured in the kidney, the lung, and the skin. The highest radioactivity concentrations were found in the liver irrespective of the dose group. In general, the concentrations determined in organs and tissues of female rats were lower than those found in male rats.

Excretion:

Excretion from the body was also a fast process: with >90 % of the given dose being excreted via urine within 48 hours. Approximately 75 % or more thereof was eliminated within the first 24 hours. The fecal elimination only played a minor role, the residual radioactivity in the body at sacrifice amounted to maximally 1 % of the given dose. Very low amounts (0.1 %) of the administered radioactivity were found in the expired air.

Metabolism:

Besides the unchanged parent compound the M06, M01, M27 and M26 metabolites were identified. The metabolites M26 and M27 consisting only of the imidazolidine ring system, could not have been detected in a study performed with the methylene-labelled test substance. However, these four metabolites were found in the urine, which contained approximately 90 % of the administered radioactivity, corresponding to an identification rate of 77 % of the recovered radioactivity.

In total, the biokinetic behavior of the imidazolidine-4,5-14C-labelled compound is very similar to the one observed with the methylene-14C-labelled compound. The administered radioactivity was rapidly and completely absorbed from the intestinal lumen and also rapidly distributed in the body. The excretion was fast and the renal route (ca. 90 % on average) was even more predominant.

 

Three further studies on the biokinetic behavior of the substance are available, with results consistent with those reported above. These will only be shortly mentioned here, for purpose of data completeness.

The distribution of the test compound, labelled with 14C in the methylene moiety was investigated in the rat by means of whole body autoradiography over a period of 48 hours following oral administration (M-024200-01-1). Further, and for the visualization of the primary distribution pattern of the radioactivity, one animal was intravenously injected and sacrificed 5 minutes after injection. The compound was administered in a single dose to male rats at a dose level of 20 mg/kg bw via both routes. The radioactivity was readily absorbed and immediately distributed to the tissues and organs of the rat. The pattern of distribution demonstrated the high ability of the radioactivity to permeate the tissues: With the exception of the fatty tissues, the central nervous system, and the mineral part of the bones blackenings on the autoradiogrammes over all other parts of the body were observed 5 minutes after intravenous injection as well as 1 hour following oral dosage. Higher concentrations were visible at later stages - except the contents of the intestinal tract - in the endocrine glands, e.g. thyroid and adrenals. The second prominent feature was the enrichment of radioactivity in the walls of the aorta and in the connective tissues of the skin. After 24 hours all other organs and tissues displayed only small amounts of radioactivity. The high degree of blackening over the kidney during the first 24 hours is a reflection of the high rate of the renal excretion of the administered radioactivity. The concentration in the fatty tissues was very low during the whole investigation period; also only small amounts of radioactivity were observed in the central nervous system. This is in good agreement with the low degree of lipophilicity of the parent compound and its metabolites and simultaneously shows that the radioactivity does not pass the blood-brain barrier very easily. With increasing time after administration the concentration of the radioactivity decreased in the organs and tissues. The relative pattern of distribution, i.e. the difference in concentration between different tissues, changed only slightly during the investigation period.

The metabolite -3-nitrosimine (M07) was identified as a minor constituent in edible plant commodities but not in the excreta of rats from studies performed according to guidelines. Since nitroso-compounds may exert adverse effects, an additional study was considered necessary, aiming to compare the test compound and its nitrosimine (M07) with regard to absorption, excretion and metabolisation (M-024174-01-1). The biokinetics of both compounds were studied at the low oral dose level of 1 mg/kg bw in the rat. For the radioactivity, the test compound as well as its nitrosamine was labelled with 14C in the methylene moiety. As far as the absorption, distribution, and excretion of the total radioactivity are concerned, no significant difference could be observed between the two compounds. The excretion ratio is in both cases approximately 3:1 (urine/feces). The corresponding nitrosamine was eliminated slightly faster from the rat's body and the organ concentrations of the total radioactivity were lower as compared to the parent compound. Thus, the reduction of the N-nitro moiety had no influence on the biokinetic behavior. In the same study, male rats were treated with a single oral dose of 150 mg/kg of the test substance. Radioactivity was excreted at the same ratio of urine to feces as after treatment with the low dose. However, the rate of excretion was significantly reduced. The metabolic pattern in the excreta was investigated after administration of a single oral dose of 1 mg/kg of the test compound and its nitrosimine to male rats as well as following the dosage of an exaggerated dose of 150 mg/kg of the test substance to male rats. The metabolites identified are in good agreement with those reported in the guideline study mentioned above (key studies). The biotransformation of the test substance to its nitrosimine (M07) was excluded under these dosage conditions. In the urine of male rats, which were orally dosed with the nitrosimine (M07), almost no metabolism was observed. Besides the unchanged nitrosimine only some 8 % of the renal radioactivity was attributable to its desnitro-compound (M09). Also in the feces some evidence for the occurrence of the latter metabolite was found. This finding demonstrates that the specific nitrosimine underwent an entirely different metabolism in comparison to its parent compound, which was all the more astonishing, since the biokinetic behavior was very similar. As a further type of investigation, the urine of rats, which have been chronically pretreated for ca. 1 year with a diet containing 1800 ppm of the test substance and additionally received one oral radioactive tracer dose of methylene-14C-labelled compound, was analyzed for the metabolites. In this test, 90 % of the total urinary radioactivity was excreted during the period of 7 to 24 hours after oral dosage. The chromatographic analysis of this urine sample revealed that 9.31 % of this radioactivity was attributable to the nitrosimine  which corresponded to 6.85 % of the administered dose. The concentration in the urine was calculated to be ca. 8 mg/100 mL. This means that the specific nitrosimine is being formed in vivo by rats under chronic feeding conditions. In order to support these result, a direct isotope dilution analysis was conducted in the urine of these chronically pretreated rats and additionally in the urine of mice, which have been fed for also approximately one year with a diet containing 2000 ppm of the test compound. Both dilution analyses clearly demonstrated the existence of its nitrosimine in the urine of either species. The concentration in the urine of rats was determined to be ca. 9 mg/100 mL, this matched the result which was obtained after administration of a radioactive tracer dose. The corresponding result in chronically pretreated mice was ca. 1.5 mg/100 mL of urine. Therefore, the findings strongly support the hypothesis that the reduction of the NO2 moiety of the test compound under formation of its nitrosimine (M07) is a biotransformation reaction in rats and mice. This obviously only takes place if enzymes catalyzing other possible reactions, e.g. the oxidative cleavage to 6-chloronicotinic acid [M14], are saturated by a chronic “flooding” of the liver with the test compound.

The metabolic behavior of the test compound was investigated in selected rat organs (i.e. liver and kidney) at different times after dosage (M-024164-01-1). The [pyridinyl-14C-methyl]-labelled test compound was orally given at 20 mg/kg in a single administration to male rats. The metabolites found in the kidney are identical with those identified in the urine. However, the triazinone-compound (M25) produced in the liver was not found in the excreta. Its formation can be explained by a non-enzymic condensation of a hypothetical amino-guanidine intermediate (M08) with pyruvic acid. This reaction was also applied in synthesizing the reference compound. The triazinone derivative probably underwent further biodegradation prior to elimination via the kidney or the bile. The relative amounts of M14 in the liver formed by oxidative mechanisms at the labelled methylene bridge remained relatively constant in the first 6 hours, possibly due to a steady state between elimination and continuous new formation from precursors, e.g. M09. In the kidney the excretory behavior of this particular organ governs the metabolite pattern in as much as the relative amount of the more polar compounds decreases with progressing while its olefine (M06) and 5-hydroxy (M01) show a relative increase at the expense of the parent compound.

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 610 mg/L at 20 °C, a molecular weight of 255.66 g/mol and a vapour pressure 4.0 x 10^-12 hPa at 20 °C. The octanol/water partition coefficient (log Pow) was determined as 0.7 at 24 °C, pH 7.

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 255.66 g/mol, a water solubility of 610 mg/L (at 20 °C) and a log Pow value of 0.7 (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 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 610 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-025697-01-1) and subacute toxicity (M-025976-01-1) dermal 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-028272-01-1) 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 4.0 x 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, acute as well as subacute toxicity studies in rodents are available. The acute inhalation study (M-027586-01-1) revealed that the test substance possesses a slight acute toxicity on uptake once via the respiratory tract, both in the form as an aerosol (high inhalability) as well as dust (relatively low inhalability). Observed systemic effects were a liver enzyme induction potential and a temporary reduction in body weight. Systemic signs of toxicity were also observed after subacute inhalation exposure of the substance (dust) to rats (M-026004-01-1). Therefore, the available data on inhalation toxicity indicate that respiratory absorption has occurred.

Overall, due to the low vapour pressure, respiratory absorption following inhalation is not considered as significant. However, after inhalation of aerosols, absorption via the respiratory tract appears to be possible, as sign of systemic toxicity was observed in rodents after inhalation exposure.

Distribution

The radioactivity was rapidly distributed from the intravascular space to the peripheral tissues and organs. At the end of the test period of 48 hours radioactivity concentrations in the tissues are very low. Levels above average are only observed in the contents of the gastrointestinal tract, liver, kidney, adrenals, thyroid, connective tissues and the vascular walls of the aorta. The extent of the penetration of the blood-brain barrier is only very limited.

Excretion

These biotransformation products and the unchanged parent compound were excreted with urine and feces, while M09 as a less important metabolite was eliminated only with the feces. The high excretion rate of the parent compound (average of 14 %) indicates a quick passage through the body, which is confirmed additionally by the elimination of more than 90 % of the recovered radioactivity within 24 hours after administration. Also from the quantitative point of view, the results are very similar. If one compares the formation of nitroimino-dehydroimidazolidine (M27) and nitroimino-imidazolidine (M26) on the one hand with 6-chloronicotinic acid (M14) its glycine conjugate (M15) and 6-methylmercaptonicotinic acid (M20) on the other hand, it can be seen that the percentage distribution in the urine is of the same order of magnitude. It has to be kept in mind that the renal excretion of the total radioactivity is higher after dosing as [imidazolidine-4,5-14C]. Those biotransformation products comprising both heterocycles, thus being directly comparable exhibit a very similar quantitative distribution pattern.

Metabolism

The metabolisation rate of the test substance in the rat is very high, it is somewhat more pronounced in male than in female animals. The main renal metabolite are 6-chloronicotinic acid (M14) and its glycine conjugate (M15) as well as the two corresponding imidazolidine ring containing biotransformation products M26 and M27. The two monohydroxylated metabolites M01 and M02 and M06 are also detected in the urine. The latter is also excreted with the feces together with M14 and M15. The amount of unchanged parent compound varied between 10 and 16 % of the given dose. Studies on the biokinetic and metabolic behavior of test compound and its nitrosimino plant metabolite (M07) in male rats yielded comparable data for absorption, distribution, and elimination. However, its nitrosimine was eliminated somewhat more rapidly, and the radioactivity levels in the organs were lower as compared to the test compound. No M07 was detected in the urine or feces following administration of a single oral doses of 1 mg/kg bw and 150 mg/kg bw of the test substance to male rats. After high doses of the test compound given to rats and mice in the diet over a one-year period, its nitrosimine was found in the urine of those animals at levels of 9 mg/100 mL (rat) and 1.5 mg/100 mL (mouse), respectively. Reduction of the nitro group of the parent compound leading to the formation its nitrosimine apparently only takes place when the enzyme systems catalyzing other possible degradation reactions are saturated. This is likely the case after chronic feeding of high test substance concentrations. The toxicological properties of its nitrosimine have thus formed a part of the results of the chronic toxicity studies in the rat and mouse. The results yield two major routes of metabolism responsible for the degradation of the test substance. At first an oxidative cleavage yielding 6-chloronicotinic acid (M14) which is conjugated with glycine forming M15. Both metabolites were found only in the urine and were excreted very quickly. These two metabolites together represented the major part of the identified metabolites with ca. 30 % of the recovered radioactivity. Only of minor importance in terms of quantity is the dechlorination of the pyridinyl moiety leading to the 6-hydroxynicotinic acid (M18) and its methylmercapto derivative (M20), probably as a degradation product of a glutathione conjugate. Possibly glutathione reacts directly with 6-chloronicotinic acid (M14) to form the same metabolite. The 6-methylmercapto nicotinic acid conjugated with glycine. The glycine conjugate (M19) amounted to 5.6 % of the recovered radioactivity. The second important biodegradation step starts with the hydroxylation of the imidazolidine ring in the 4- or 5-position. About 16 % of the recovered radioactivity was identified as the sum of 4- and 5-hydroxy-imidacloprid (M01, M02). The loss of water yields M06.

 

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.