Teflubenzuron

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1985-08-26 to 1986-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Objective of study:

absorption

excretion

metabolism

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

EC Number:

617-441-5

Cas Number:

83121-18-0

Molecular formula:

C14 H6 Cl2 F4 N2 O2

IUPAC Name:

1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea

Test material form:

solid: crystalline

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

rat

Strain:

other: Chbb: THOM (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Boehringer Ingelheim KG, Germany
- Age at study initiation:
Excretion tests: 41 - 49 days (male rats), 51 - 67 days (female rats)
Blood level tests: 46 - 54 days (male rats), 59 - 85 days (female rats
- Weight at study initiation:
Excretion tests: 175 - 225 g
Blood level tests: 200 - 250 g
- Housing: Individually in metabolism cages
- Diet: ad libitum , commercial diet "Altromin 8013", Altromin GmbH, Germany
- Water: ad libitum, tap water
- Acclimation period: at least 2 days
- Health status: only healthy animals were used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
- Fasting period: Animals were fasted 14 hours prior to dosing to 2 hours post treatment (test groups B - F). Thereafter each animal had access to 25 g diet daily. The animals of test group A were supplied with food immediately after treatment inorder to avoid loss of volatile radioactivity in case of later feeding.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: The test substance was applied as a suspension in a mixture of 1 % Tylose C 30 (methylcellulose) and 1 % Tween 80 (1:1).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For each application date, the suspension was prepared separately as follows: The amount of parent compound needed at the respective application day was given into a mortar of a high speed micro mill (Spectro-Mill MS). After adding an aliquot of the suspension mixture, the active ingredient was milled over a period of ten minutes. Thereafter the milled test substance was washed into a suitable flask using the rest of the needed suspension mixture. The actual radioactivity content of each application mixture was determined by liquid scintillation counting. At the low dose level, each animal received 0.5 mL of the application suspension. At the high dose level, 1.0 mL was applied to each animal.

Duration and frequency of treatment / exposure:

Groups A, B and E: These animals each received a single oral dose of the radiolabeled test substance at the low dose level.
Group C: These animals each received a series of daily doses of the non-labelled test substance over a period of 14 days, followed at 24 hours after the last dose by a single oral dose of the 14C-labelled test substance. Each dose was at the low dose level.
Groups D and F: These animals each received a single oral dose of the radiolabeled test substance at the high dose level.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

A) 1 male, 1 female; single low dose; test on expired air
B) 5 males, 5 females; single low dose; excretion and metabolism
C) 5 males, 5 females; repeated low dose; excretion and metabolism
D) 5 males, 5 females; single high dose; excretion and metabolism
E) 5 males, 5 females; single low dose; blood level
F) 5 males, 5 females; single high dose; blood level

Control animals:

no

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale:
The low dose should correspond to a no-effect level. The high dose should produce toxic or pharmacologic effects. Due to the extremely low toxicity of the substance it is not possible to produce toxic effects with the highest amount of substance which can be administered without difficulties by gavage. For this reason this highest amount which can be applied was chosen as high dose level. This corresponded to 750 mg test substance/ kg bw.
Starting from this, the 30-fold lower dose was chosen as low dose level in order to gain unequivocal information on the rate of absorption in relation to the administered dose. On the other hand, isolation and identification of individual metabolites exceeding 1 % of dose was still possible. In addition, this low dose level corresponded to the dose level used in the preliminary tests on the biokinetics and metabolism in the rat. In these tests the compound was administered solubilized in dimethyl sulfoxide. By choosing the same dose for the present test, it was possible to gain additional information on the extent of absorption of the test substance from the gastro-intestinal tract of the rat. According to these considerations the following low dose level was used: 25 mg test substance/ kg bw. The amounts of test substance actually applied to each rat were determined by weighing the (filled) application syringes prior to and after treatment.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
Group A:
Two rats (1 male and 1 female) were dosed with the radiolabelled test substance at the low dose level and then placed in all-glass metabolism cages suitable for the collection of expired air. After application of the test substance the rats were transferred to the metabolism cages. They were supplied with food and water immediately after treatment in order to avoid loss of volatile radioactivity in case of subsequent feeding. For a period of 24 hours a constant stream of air (flow rate approx. 230 cm3/min) was drawn through the devices by means of water Jet pumps.
Groups B, C and D:
Ten rats from each group were dosed and then transferred to metabolism cages suitable for the separate collection of urine and feces. Both, urine and feces were collected at the following times after application of the radiolabeled test substance: 24, 48, 72, 96, 120, 144, 168 and 192 hours. After completion of the excretion period the animals were killed by exsanguination whilst under chloroform anesthesia.
Groups E and F:
Ten rats (5 male and 5 female) from each group were dosed and then transferred to individual metabolism cages. Serial blood samples were taken from the postorbital venous plexus of each rat at the following time intervals: before treatment and 20, 40, 60 minutes, 2, 4, 6, 8, 24, 48, 72, 96, 120 and 168 hours after treatment. During blood sampling each animal was kept under short halothane anesthesia.


ANALYTICAL METHOD
Group A:
The absorption solution of each absorption tower (70 mL) was separately filled into a 100 mL volumetric flask. The radioactivity content of each flask was determined by liquid scintillation counting.
Groups B, C and D (urine):
Urine was transferred to 100 mL volumetric flasks. Urine sampling devices were washed. The wash solutions were added and the flasks made up to the mark with deionized water. Subsequently, the radioactivity of each urine sample was determined by liquid scintillation counting. Thereafter, the urine samples were pooled by day and sex and stored deep frozen till analysis. After completion of each test, the cages were washed using acetone, methanol and water (30 mL each). The wash solutions of each cage were combined and investigated for their radioactivity content. The following amounts of urine of the first day after treatment were used for thin-layer chromatographic investigations:
Test groups B and C: Total urine of each sex
Test group D: Aliquots of 200 mL of each sex.
The samples were freeze-dried and subsequently extracted with 2 x 100 mL acetone and 2 x 100 mL methanol. Finally, the residues were dissolved in a sufficient amount of water (50 - 100 mL). The solutions were combined according to extraction solvents and reduced in volume using a rotary film evaporator under reduced pressure. Solutions containing sufficient amounts of radioactivity were directly applied to thin-layer plates. If sufficient amounts of radioactivity were present, possible conjugates contained in the polar metabolite fractions of the urine were further investigated as follows:
Enzyme treatment: Portions of polar metabolite fractions contained in urine were separated by TLC, dissolved in 1 mL acetate buffer (pH 3.5) and incubated with 0.5 mL beta-glucuronidase/arylsulfatase The incubation period was approx. 12 hours at 37 °C after which the samples were evaporated to dryness using a rotary film evaporator. The solid residues were extracted three times with 10 mL acetone. The remainder was finally dissolved in 10 mL methanol/water (4:1). Each extraction step was carried out with 10 min ultrasonic treatment. The acetone solutions were combined and reduced to a total volume of 10 mL. Subsequently, solutions containing sufficient radioactivity were investigated by TLC.
Treatment with hydrochloric acid: Portions of polar metabolite fractions contained in urine were separated by TLC, dissolved in 2 mL 2N hydrochloric acid and incubated at 40 °C over a period of approx. 20 hours (the test substance proved to be stable under these conditions as demonstrated with the authentic parent compound). Thereafter the samples were neutralized with 2N sodium hydroxide solution and evaporated to dryness using a rotary film evaporator. The solid residues were extracted three times with 10 mL acetone. If still necessary, the remainder was finally dissolved in 10 mL methanol. Each extraction step was carried out with 10 min ultrasonic treatment. The acetone solutions were combined and reduced to a total volume of 10 mL. Subsequently, solutions containing sufficient amounts of radioactivity were investigated by TLC.
Groups B, C and D (feces):
After determination of fresh weight, feces were freeze-dried and subsequently homogenized in a mortar. Thereafter the following fecal samples were successively extracted with 2 x 50 mL acetone and 2 x 50 mL methanol/ water (4:1):
Test groups B and C: Samples of the first three days after treatment.
Test group D: Samples of the first four days after treatment
The extraction of the remaining fecal samples was omitted because chromatographic investigation was no longer possible with the low amounts of radioactivity still extractable. Each extraction step was carried out with 10 min ultrasonic treatment. Finally, the extracted fecal samples were additionally washed with 50 mL acetone in order to remove water remnants and subsequently air-dried. The wash solution was added to the last methanol/water extract. The extracts of each fecal sample were combined according to extractants, reduced by means of a rotary evaporator and transferred to 50 mL or 100 mL volumetric flasks. Tests with selected samples proved that no loss of radioactivity occurred during reduction of volume. The radioactive content of each extract was determined by liquid scintillation counting. Extracts containing sufficient amounts of radioactivity were subjected to radio thin-layer chromatography. Depending on the amounts of co-extracted impurities, the limits of possible TLC-evaluation were in the range of approx. 500- 1000 dpm per mL of solution. After weight determination, the extracted and air-dried fecal residues of test groups B and D were subjected to combustion analysis. For this purpose, three aliquots of approx. 100 mg of each sample were combusted in the sample oxidizer. All extracted fecal samples of test groups B and D containing 2 1 % non-extractable radioactive residues (related to the applied radioactivity) were additionally subjected to a more severe extraction procedure. According to the results found in groups B and D, the extracted fecal samples of the two first days of group C were subjected to the same additional extraction procedure prior to combustion analysis. Extracted fecal samples of group C originating from day three were analyzed by combustion analysis without additional extraction. Additional extraction procedure: Amounts of 100 mL methanol/water (4:1) were added to each fecal sample. Subsequently, the samples were heated to reflux for 30 min. Following filtration and washing with 50 mL of the same solvent mixture, the combined solutions were reduced in volume by means of a rotary evaporator and transferred to a 50 mL volumetric flask. Thereafter the radioactivity of each solution was determined by liquid scintillation counting. Extracts which contained sufficient radioactivity were subjected to radio thin-layer chromatography. Fecal samples which had been treated in this way were subsequently analyzed by combustion analysis in order to determine the radioactive residues still non-extractable. The radioactive residues contained in non-extracted fecal samples (test groups B and C: sampling days 4-8, test group D: sampling days 5-8) were determined by combustion analysis, too.
Blood samples of test groups E and F:
At each sampling time, blood samples (approx. 200 - 300 mg blood) were taken from each rat by means of heparinized micro hematocrit tubes. Each sample was transferred to a sealable centrifuge tube and centrifuged immediately for a period of 5 min at 5000 rpm. Subsequently, the exact amount of blood was determined by weighing. The supernatant plasma was separated by means of a syringe, filled into a scintillation vial and weight. Thereafter, the radioactivity of the plasma samples was determined by liquid scintillation counting. The red cells which remained in the centrifuge tube, were mixed with 0.5 mL 0.1N sodium hydroxide solution and transferred quantitatively to a scintillation vial. Herein, the sample was decolorized and solubilized by adding 0.5 mL H202 and 0.4 mL Digestin. Subsequently, the radioactivity was determined by liquid scintillation counting.
Residues in rats carcasses (test groups B, C and D):
The carcasses of the rats were solubilized by adding 1 ltr. 2N sodium hydroxide solution and heating to 50 °C over a period of 4 days. Subsequently, the supernatant soap originating from the body fat was separated, dissolved in water and filled into 250 mL volumetric flasks. The amounts of radioactivity contained in the alkaline solution and in the soap solution were determined by liquid scintillation counting.

Measurement of radioactivity:
Aliquot samples of faecal extracts, urine and other liquid samples were mixed with 15 ml scintillation mixture ("Quickszint 212", W. Zinsser Co., D-6000 Frankfurt, Fed. Rep. of Germany).
In case of plasma, each total sample was used for determination of radioactivity.
Red cells of blood were decolorized and solubilized as discribed.
Aliquots of solid samples were burned in oxygen using an automatic sample oxidizer. Combustion products were absorbed into an appropriate scintillation mixture (675 ml ethanolamine, 825 ml toluene, 1350 ml methanol, 18 g 2,5-diphenyloxazole [PPO]). The efficiency of the oxidizer was checked using carbon-14 standards.
Radioactivity was measured by liquid scintillation counting (LSC). The scintillation counters were calibrated by automatic external standard quench parameter standardisation. The accuracy of the scintillation counters were checked daily.
The validity of the calibration curves were checked at intervalls of one week. For all performance verification tests commercially prepared quenched and unquenched standards were used (Packard Instrument Company, Inc., Downers Grove, Ill. 60515, USA).
All samples were counted twice.
Counting of blood samples (plasma, solubilized red cells) was terminated when the statistical 20 error of the counting rate reached 5 % or after 30 minutes, whichever came first.
Counting of all other samples was usually pre-set at 10 minutes. Selected samples containing extremely low radioactivity were counted over periods of 30 minutes.
Amounts of radioactivity of less than twice the background levels (approximately 20-40 cpm depending on scintillation mixture and scintillation counter used) were considered to be below the limit of accurate measurement.

Thin-layer chromatography (TLC):
One-dimensional thin-layer chromatography on precoated plates of silica gel 60, F 254 (Merck Co., D-6100 Darmstadt, Fed. Rep. of Germany) was carried out using the following solvent systems (with chamber saturation):
system 1: ethyl acetate/cyclohexane 1 : 1
system 2 : ethyl acetate/cyclohexane 3 : 7
system 3: dichloromethane
system 4 ' ethyl acetate/cyclohexane 7 : 3
System 5: diisopropyl ether/acetone 7 : 3
Unlabelled test substance and possible degradation products were also applied to the thinlayer
plates for comparison. The chromatograms were evaluated using the TLC linear analyzers. The results were processed via programmable on-line calculators (HP-97 S, Hewlett Packard).

High pressure liquid chromatography (HPLC):
High pressure liquid chromatography was performed using the following conditions:
Column: Hibar LiChroCART 125-4 HPLC—cartridge (Merck Co., D-6100 Darmstadt, Germany)
stat. phase: LiChrosorb RP-18
length: 125.00 mm
internal diameter: 4.00 mm
particle size: 7 µm
Eluent: acetonitrile/water 65:35
Flow rate: 1.0 ml/min or 0.5 ml/min
UV-detection: Photo diode array detector
Detection of radioactivity: Collection of fractions of column eluate and measurement of their radioactivity by liquid scintillation counting.

UV-spectroscopy:
UV-spectra of the parent compound and of the metabolites were recorded using an HPLC UV-photodiode array detector. The compounds were introduced via the HPLC-system. The UV spectra
were processed using an IBM XT Personal Computer.

Mass-spectroscopy:
Electron impact mass spectra were performed by Boehringer Ingelheim KG using a Varian-MAT-CH7 mass spectrometer.
Conditions: Direct introduction
Temperature of ion source: 200°C
Energy of ionization beam: 70 eV
Trap current: 300 uA

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

poor absorption (4-10%)

Main ADME results 2

Type:

excretion

Results:

main route of excretion via feces (>85 %) excreted within 24 h after treatment, greatest portion excreted with feces was unchanged parent; only 0.1-0.9 % found in urine; no differences in excretion pattern found between single and repeated dose treatment

Main ADME results 3

Type:

metabolism

Results:

no differences between sexes, greatest portion of substance excreted unchanged (82-91%); trace amounts of metabolites consisting essentially of polar substances, one of them 3,5-dichloro-2,4-difluorophenylurea in feces

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Test group E (single low dose):
The radioactivity concentrations in the plasma of male and female rats found after an oral administration of 25 mg/kg bodyweight are presented. Twenty minutes after treatment, plasma concentrations of 0.16 µg equivalents test item/mL (male) and 0.14 equivalents/mL (female) were found. During the first hour of the test, the values slightly increased up to 0.38 µg/mL in the males and 0.25 µg/mL in the females. From the first to the eighth hour post treatment, nearly constant plasma levels were found (males: 0.38 - 0.46 µg equivalents/mL, females: 0.22 - 0.25 µg equivalents/mL). The plasma levels already declined 24 hours after treatment (male rats: 0.24 µg/mL, female rats: 0.17 µg/mL) and were less than 0.01 µg/mL in both sexes at the end of the experimental period (7 days).
The concentrations of radioactivity present in the whole blood were comparable to those found in the plasma. In the 1-8 hours blood samples of male rats, 0.25 - 0.36 µg equivalents/mL were found. The corresponding values of female rats amounted to 0.18 - 0.28 equivalents/mL. Up to the seventh day of the test, the radioactivity concentrations in the whole blood declined to 0.02 µg/mL and 0.01 µg/mL in male and female rats, respectively.

Test group F (single high dose):
The radioactivity concentrations in the plasma of male and female rats obtained after an oral dose of 750 mg/kg bodyweight are presented. Following the application of the high dose, the plasma radioactivity concentrations were higher in both sexes when compared to the results found after application of the low dose. Nearly constant plasma levels were found in the male rats during a period between 40 minutes and 8 hours post treatment. During this time, the plasma levels amounted to 1.17 - 1.72 µg equivalents/mL plasma. Twenty-four hours after treatment a peak mean concentration of 3.27 µg/mL occurred in males.
Nearly constant plasma levels were found in female rats too, lasting over a period of eight hours. In this case, the plasma levels amounted to 0.98 - 1.43 µg equivalents/mL plasma during the first eight hours of the test. A peak concentration comparable to that found in the male animals was not observed. It cannot be excluded, however, that a comparable maximum value occurred between the eighth and the twenty-fourth hour in female animals, too.
From the second day after treatment the plasma levels declined reaching 0.06 and 0.08 µg/mL at the end of the test (7 days) in male and female animals, respectively.
The values of radioactivity present in the whole blood were slightly lower when compared with the plasma levels. Nearly constant blood levels were reached over periods of time comparable to those outlined above. During these periods, values in the range of 0.78 - 1.22 µg equivalents/mL were observed in the male rats. The peak value at 24 hours amounted to 2.51 µg/mL. In female rats, values in the range of 0.67 - 0.96 µg equivalents/mL were found. The concentrations in the whole blood of male and female animals declined to 0.35 µg/mL and 0.13 µg/mL, respectively, till the end of the test (7 days).

It can be concluded that the absorption of test item from the gastro-intestinal tract of the rat does not exceed 10 % of the dose if 25 mg active ingredient/kg bodyweight are administered in suspension. After administration of 750 mg active ingredient/kg bodyweight, a maximum absorption rate of just 4 % of the dose can be expected.

Details on distribution in tissues:

not assessed in this study.

Details on excretion:

Test group A (expired air):
No radioactivity was detected in the expired air during 24 hours after administration of 14C-labeled substance, demonstrating that the radioactivity was situated in metabolically stable positions (radioactivity applied: 11.4 x 10E8 dpm, limit of detection: 13 dpm = 0.0001 % of the dose).

Test group B (single low dose):
The excretion patterns of radioactivity found after oral administration of the low dose (25 mg/kg bw) of 14C-labeled substance are described for male and female rats. The results reveal that the main excretion route of radioactivity was via the feces. Furthermore, the compound was excreted almost completely within a short period of time. All animals excreted > 85 % of the dose within 24 hours post treatment. A mean excretion rate of more than 90 % was achieved during the second day of the test. Only 0.05 % of the radioactivity applied could still be detected in the carcasses of rats at the end of the test (eight days after treatment). 0.86 % (males) and 0.55 % (females) of the radioactivity were excreted in the urine. During the same period, means of 93.3 % and 90.7 % were found in the feces of male and female animals, respectively. The total recovery of the radioactivity applied was 94.2 % (males) and 91.3 % (females).

Test group C (repeated low dose)
The excretion patterns obtained after 14 daily treatments with unlabeled substance and one subsequent treatment with the 14C-labelled compound (low dose: 25 mg/kg bw each) are presented for both sexes. The results of this test group reveal no significant difference between the excretion patterns found after single and repeated treatment. After repeated treatment the compound was also completely excreted within a short period of time. In the majority of the animals showing normal defecation, the 90 % excretion was reached after 24-48 hours . The radioactivity contained in the carcasses amounted to 0.04 % (males) and 0.08 % (females), only, at the end of the test (8 days after administration of labelled substance). The feces again contained very high levels of radioactivity (males: 94.0 %, females: 94.9 % of the applied radioactivity). In the urine 0.84 % and 0.78 % were found. Means of 94.9 % and 95.7 % of the administered dose were recovered from male and female rats, respectively.

Test group D (single high dose):
In this test group, an extremely low recovery of radioactivity was found in one male rat. Evaluation of all data of indicates that parts of the application mixture must have been spat out immediately after treatment. At that time, the animal was still kept in a tray outside the metabolism case. By this way the animals fur and (subsequently) the metabolism cage was contaminated. Consequently, the outlying results found in this rat ware excluded from the calculation of mean excretion and retention rates. The excretion patterns of radioactivity obtained after oral administration of the high dose (750 mg/kg bw) of 14C-labled substance are presented. The results reveal that the radioactivity was also excreted completely within a short period of time after application of the high dose. More than 90 % of the dose were also excreted in this test group within 24 - 48 hours. The radioactive residues found eight days after treatment in the carcasses of rats amounted to less than 0.01 % of dose in both sexes. 95.0 % (males) and 93.6 % (females) of the radioactivity applied were excreted in the feces. The amount of radioactivity excreted in the urine was 0.15 % (males) and 0.16 % (females), only. The total recovery amounted to 95.1 % and 93.7 % in the male and female animals, respectively.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The TLC comparison of the extracts revealed that in all test groups by far the greatest portion of radioactivity excreted in the feces was the unchanged parent compound itself. This was confirmed by mass-spectroscopic investigation of the radioactive compounds isolated from selected extracts. The identity of the unchanged parent compound excreted in the feces was further confirmed by HPLC and UV-spectroscopy.
As a consequence of the high recovery rates of the parent compound, only small amounts of radioactive metabolites were found in the fecal extracts:
Test group B, male rats: 6.5% of dose, female rats: 5.9% of dose
Test group C, male rats: 5.5% of dose, female rats: 4.8% of dose
Test group D, male rats: 2.8% of dose, female rats: 3.0% of dose
These portions of radioactivity were composed of trace amounts of different products contained in the diverse extracts. One of these products could be identified as 3,5-dichloro-2,4-difluorophenylurea. The metabolite was found in the feces of some rats of group D (high dose level) after extraction with methanol/water (4:1) in the heat. In this extract the metabolite amounted to 0.02-0.03 % of dose in male and female rats, respectively. The possibility of artificial cleavage of test item, as a result of the treatment, can be excluded. Parent compound, which was added to feces of rats and treated in the same way, proved to be stable. The metabolite was identified by comparison with the authentic compound using TLC, HPLC and UV-spectroscopy. Mass spectroscopic investigation of the metabolite was not possible due to the low amount of substance and the relatively high amounts of impurities still present after the comprehensive isolation procedure. The total amount of this fecal metabolite was calculated by adding the traces of radioactivity which co-chromatographed with the authentic compound in all fecal extracts. From this calculation follows that the metabolite by no means exceeded 0.2 % of dose in both sexes after application of the high dose. Traces of the metabolite could be detected by TLC in the fecal extracts of some rats of test groups B and C (low dose level), too. From the amounts of radioactivity which co-chromatographed with the authentic compound, it can be concluded, that in these test groups the metabolite by no means exceeded 0.5-1.0 % of dose. Further confirmation of the identity using other methods was not possible in this case due to the very low amounts of substance.
3,5-dichloro-2,4-difluoroaniline, the second possible metabolite which was available as authentic compound, could not be identified definitely in the fecal extracts. From trace amounts of radioactivity, which co-chromatographed with the reference substance, it can be excluded that this possible metabolite; if present at all, would exceed 0.4-0.7 % of dose after administration of the low dose level. In the case of the high dose level this possible metabolite would by no means exceed 0.1-0.2 % of dose. Further identification of the unknown polar metabolites contained in the fecal extracts was not possible due to the extremely low amount of each individual substance and the huge surplus of co-extracted fecal components.
In the acetone extract of the urine of group D traces of the unchanged parent compound were found by thin-layer chromatographic comparison with the authentic active ingredient. This could be confirmed by HPLC and UV-spectroscopy. The total amount of the parent compound found in the urine of male and female rats after treatment with the high dose was about 0.01-0.02 % of the radioactivity applied. It cannot be excluded, however, that these extremely low amounts of unchanged substance resulted from small particles of feces the total separation of which is impossible.
From the thin-layer chromatogram the presence of the fecal metabolite 3,5-dichloro-2,4-difluorophenyl-urea could be presumed in the urine, too. HPLC-investigations of the isolated TLC-fraction (< 0.01 % of dose only) revealed the presence of very small amounts of a compound showing the same retention time as this metabolite. In addition, a large surplus of several impurities was found by HPLC. Further confirmation of the identity of the metabolite by UV-spectroscopy or other methods was consequently not possible. The polar compounds remaining at the origin of the TLC-plates were further treated as follows: radioactivity contained in zone 1 of the thin-layer chromatograms of the acetone extracts was isolated. The isolated fractions as well as the radioactive compounds contained in the methanol extracts were subsequently treated with both hydrochloric acid and B-glucuronidase/arylsulfatase. TLC of the cleavage products were attempted. From the comparison of these chromatograms with those obtained prior to treatment, it can be seen that at least parts of the polar metabolite fractions proved to have been conjugates. The radio chromatograms could only be obtained after extremely enlarged counting times. This was caused by the fact that large amounts of impurities (urinary compounds, enzymes, buffer components, salts resulting from neutralization) were contained in the individual solutions. Furthermore, an inevitable fractionation of the radioactivity originally contained in the urine resulted from the different extraction steps which followed freeze drying and cleavage procedures. Consequently, only extremely low trace amounts of radioactivity could be applied to the thin-layer plates. Further identification of any cleavage product was impossible, too, due to the very low amount of each compound and the huge surplus of impurities.

From the results obtained from all studies, it can finally be concluded that the substance is metabolized in the rat by cleavage of the urea bridge as well as by hydroxylation of one of the phenyl rings. In the latter case one fluorine atom is substituted if hydroxylation takes place in the aniline ring. Both routes of metabolization, however, yielded only trace amounts of metabolites due to the extremely low absorption rate in the rat.

Any other information on results incl. tables

Table 1: Concentration of radioactivity in the feces, urine and carcass up to eight days post-administration. Expressed as % of the administered dose.

FECES
	MALES	FEMALES
Single low dose (25 mg/kg bw)	93.3 %	90.7 %
14 repeated low dose (25 mg/kg bw) of cold material + 1 single low dose of^14C-test item	94.0 %	94.9 %
Single high dose (750 mg/kg bw)	95.0%	93.6%
URINE
Single low dose (25 mg/kg bw)	0.86 %	0.55 %
Single repeated low dose (25 mg/kg bw) of cold material + 1 single low dose of^14C-test item	0.84 %	0.78 %
Single high dose (750 mg/kg bw)	0.15 %	0.16 %