(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987 - 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliable study conducted to fulfill data requirements of EPA guideline 85-1 and following GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Objective of study:

absorption

distribution

excretion

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

(1982)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

EC Number:

406-850-2

EC Name:

(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

Cas Number:

133855-98-8

Molecular formula:

C17 H13 Cl F N3 O

IUPAC Name:

1-{[(2R,3R)-3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazole

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Recognized by international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH-4414 Fuellinsdorf/Switzerland
- Weight at study initiation: The body weight was assessed 1 to 3 days prior treatment with 14C-labeled test substance and ranged from 144-226 g.
- Fasting period before study: Rats were fasted overnight prior dosing, except for rats submitted to repeated treatments and after bile cannulation.
- Housing: In groups of 2-3 rats in Makrolon cages
- Individual metabolism cages: yes, if appropriate.
- Diet ad libitum: Pelleted 343-Kliba rat maintenance diet
- Water ad libitum: Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8 - 25.1°C
- Humidity (%): 34-88%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

other: Depending on Experiment via oral gavage or via feeding

Vehicle:

acetone

Remarks:

(oral gavage)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Overall 5 stock solutions were prepared by mixing the labelled test substance (specific radioactivity: 180.92 mCi/g) with non-labelled test substance in acetone and stored at -20°C. The stock solutions used at the high dose level showed a specific radioactivity of 16.90mCi/g (stock solution I), 6.22mCi/g (stock solution II) and 5.91mCi/g (stock solution III). The stock solutions used at the low dose level showed a specific radioactivity of 40.0mCi/g (stock solution I) and 39.71mCi/g (stock solution V).
PREPARATION OF FORMULATIONS
Based on the intended dose level and body weight of the rats, appropriate aliquots were taken. After storage at -20 °C the amount of labeled test substance in the respective stock solution and its specific weight were re-determined. In one case the remaining turbid stock solution (2.5 ml) had to be diluted with acetone to obtain a clear stock solution.
Aliquots of 100 µl per 100 g bw were mixed with about 80 mg CREMOPHOR EL (BASF, Ludwigshafen) and 0.9-1.18 ml 1 % MC 4000 in 0.9 % NaCl and stored at -20 °C until administration. The total aliquot was administered at once. If appropriate, the application vial was rinsed and re-administered to increase the recovery. After administration, the application devices were washed with acetone and the radioactivity was determined by liquid scintillation counting. By this procedure, the amount actually administered to each rat was calculated.
PREPARATION OF DIET
Labelled food
Based on the average food consumption during 14 days and the intended dose level of 3 mg/kg, an aliquot of the stock solution was mixed with acetone and 400 g granulated food by means of a Hobart mixer, resulting in a concentration of about 41 mg/kg of diet. After addition of bidistilled water, food was prepared by means of a meat-mincer. The pieces were allowed to dry and were thereafter stored in daily aliquots at -20 °C until use.
Non-labelled food
Based on the average food consumption of 10 - 13.0 g untreated food per 100 g rat body weight during 5 days before treatment and the intended dose level of 3 mg/kg, non-labelled test substance was dissolved in 250 ml acetone and thoroughly mixed with 2.55 kg granulated food. Thereafter, daily aliquots were prepared as described for the labelled food. Three aliquots of 20 g were separated for HPLC-analysis (see below).
For the excretion-experiment a batch of the non-labelled test substance was prepared for each week. The formulation of the non-labelled test article was as described for the 14C-labelled test substance. The batches were divided in daily aliquots based on the dose level of 3 mg/kg and the body weight of each rat and stored at -20 °C. Before administration, aliquots were adapted to room temperature. No HPLC-analysis was performed.
CONCENTRATION OF TEST MATERIAL
The pulverised food was extracted with water/ethanol (1/7). An aliquot of the non-labelled food was evaporated and the residue was re-dissolved in 20 µl water/acetonitrile (3/7) by means of ultrasonic treatment. An aliquot was injected onto an HPLC-column (Lichrosorb RP18 12.5 cm, flow rate of 0.5 ml/min, UV-detection at 210 nm). The concentration of non-labelled test substance in food was 23 mg/kg diet. The concentration of the labelled test article was determined by combustion followed by liquid scintillation counting (LSC).

Duration and frequency of treatment / exposure:

The study consisted of 6 experiments:
Experiment I (Pre-test): single oral gavage; 48 h recovery period
Experiment II (Balance study): single oral gavage; 168 h recovery period
Experiment III (Tissue distribution): either single oral gavage (100 mg/kg bw) or repeated (7x) daily oral gavage (3 mg/kg bw); 2, 24, 48, 96, 168 h recovery period
Experiment IV-Part I (Blood level): single oral gavage; 168 h recovery period (blood samples were taken at different time points)
Experiment IV-Part II (Blood level): 14 days feeding study with non-labelled test substance followed by 3 days feeding study with radio-labelled test substance; No recovery period
Experiment V (Excretion via bile, urine/feces): single oral gavage; 15 – 48 h recovery period (depending on health status and excretion pattern)
Experiment VI (Excretion via urine/feces): repeated 14 day oral gavage of non-labelled test substance followed by a single oral gavage 14C-labelled test substance; 168 h recovery period

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose / concentration:

The study consisted of 6 experiments:
Experiment I (Pre-test): 2 males and 2 females per dose
Experiment II (Balance study): 5 males and 5 females per dose
Experiment III (Tissue distribution): 20 males and 20 females overall; 2 males and 2 females per time point and dose
Experiment IV-Part I (Blood level): 10 males and 10 females (splitted into 4 groups á 5 animals) per dose
Experiment IV-Part II (Blood level): 5 males and 5 females per dose
Experiment V (Excretion via bile, urine/feces): 4 males and 3 males at 3 mg/kg bw; 3 males and 4 females at 100 mg/kg bw
Experiment VI (Excretion via urine/feces): 5 males and 5 females per dose

Control animals:

no

Positive control reference chemical:

not included

Details on study design:

- Dose selection rationale:
In a pre-test no mortality was seen within 48 h after oral application of 100 mg/kg bw test substance to 2 males and 2 females via gavage. Therefore, 100 mg/kg bw was used in this study as high dose level.

Details on dosing and sampling:

STUDY DESIGN (an overview of the study design is given in any other information on materials and methods)
The study consisted of 6 experiments. The design of the experiments was as follows:
Experiment I (Pre-Test):
Experiment I was conducted as a Pre-test to determine mortality at the high dose level (100 mg/kg bw) within 48 h after oral gavage of 100 mg/kg bw test substance to 2 males and 2 females.
Experiment II (Balance study after single oral administration of 14C-labelled test substance):
This experiment consisted of two sub-experiments. First, 5 male rats were treated with 100 mg/kg bw radio-labelled test substance and the radioactivity in the expired air, urine, feces, organs/tissues, residual carcass and digestive tract were determined after recovery (168 h).
In the second sub-experiment, 5 female and 5 male rats received 3 mg/kg bw labelled test substance per gavage. In addition, 5 female rats received 100 mg/kg bw labelled test substance. The radioactivity in urine, feces, organs/tissues, residual carcass and digestive tract were determined after recovery (168 h).
Experiment III (Bio-retention study after single and repeated oral (7x) administration of 14C-labelled test substance):
Male and female rats were either given a single oral gavage of 100 mg/kg bw or a repeated (7x) daily oral gavage of 3 mg/kg bw/d of the 14C-labelled test substance, respectively. The tissue distribution of radioactivity was investigated 2, 24, 48, 96 and 168 h after treatment (2 animals of each sex per time point).
Experiment IV (Blood level of radioactivity after single and repeated oral gavage of 14C-labelled test substance):
This experiment consisted of two main experiments (IV-I and IV-II). In the first experiment, radioactivity in blood and plasma was determined in male and female rats after single oral administration of the 14C-labelled test article at 3 and 100 mg/kg bw. Blood was withdrawn retro-orbitally at 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144 and 168 h (Experiment IV-Ia).
The amount of the parent compound in blood was assessed in males and females after treatment with 3 and 100 mg/kg bw14C-labelled test substance, respectively by single gavage. Blood was withdrawn retro-orbitally at 2, 24, 96 and 168 h after administration into heparinised containers. The blood samples were pooled (per time interval, sex and dose level) and were stored at -20 °C until analysis (Experiment IV-Ib).
In the second part of this experiment (IV-II), radioactivity in plasma was determined in male and female rats after a 14 days feeding period with food containing non- labeled test substance (target dose: 3 mg/kg bw/d) followed by a 3 days feeding period with food containing radio-labelled test substance (target dose: 3 mg/kg bw/d). Blood was collected from the retro-orbital sinus into heparinised containers on day 15, 16 and 17. The food consumption was recorded daily.
Experiment V (Biliary excretion of radioactivity after single oral administration):
The radioactivity in bile, urine and feces, as well as radioactivity in carcass and digestive tract was assessed in male and female rats after single oral gavage of 3 and 100 mg/kg bw 14C-labelled test substance, respectively. The recovery period ranged between 15 and 48 h (depending on health status and excretion pattern). Since <0.1 % of the radioactivity administered were detected in plaster and bandage of the high dosed females after extraction with acetone the radioactivity of the plasters and bandage were not investigated for the other groups.
Experiment VI (Excretion of radioactivity after repeated oral (14x) administration of non-labelled test substance followed by a single oral administration of labeled test substance):
The radioactivity in urine and feces of male and female rats was assessed upon oral gavage of non-labelled test substance for 14 days (3 mg/kg bw) and a final oral gavage with the 14C-labelled test substance (3 mg/kg bw). Radioactivity of urine, feces, organs/tissues, blood, intestinal tract, residual carcass and cage wash was determined after a 168 h recovery period.

MEASUREMENT OF RADIOACTIVITY
Radioactivity was determined with Packard liquid scintillation counters equipped with DPM and Luminescence options (Tri-Carb 460 CD or 2000 CA). All measurements, except for plasma and blood samples from the balance studies (Experiment II)and organs/tissues, were corrected for background. Measurements were performed for a counting time allowing a counting accuracy between 5 and 10%.

SAMPLING METHODS/DETERMINATION OF RADIOACTIVITY
EXPIRED AIR: The glass metabolism cages were ventilated (600 ml air/min) and the outcoming air was passed through a trapping system. The absorption solution of the trapping system was changed 8, 24, 32 and 48 h after treatment.
URINE/FECES: Urine was collected in 24 hour intervals until 168 h after the administration into dry-ice/ethanol cooled tubes. Feces were collected accordingly at room temperature. Volumes and fresh weights were noted. Radioactivity in urine was determined by liquid scintillation counting of subsamples. Feces were homogenized in the presence of an equal volume of water and the radioactivity was determined by combustion of subsamples.
BLOOD/PLASMA: Animals were anesthetized with carbon dioxide and sacrificed by exsanguination 168 h after treatment. Blood was collected into heparinised tubes, aliquots were separated and plasma was generated via centrifugation.
To determine radioactivity in blood at different time points (Experiment IV) blood was collected from the retro-orbital sinus at 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, 144 and 168 h (Experiment IV-Ia) and at 2, 24, 96 and 168 h after administration into heparinised containers.
The radioactivity in plasma in male and female rats after a 14 days feeding period with food containing non- labeled test substance (target dose: 3 mg/kg bw/d) followed by a 3 days feeding period with food containing radio-labelled test substance (target dose: 3 mg/kg bw/d) blood was collected from the retro-orbital sinus into heparinised containers on day 15, 16 and 17(Experiment IV-II).
The radioactivity in blood and plasma was determined after digestion with tissue solubilizer.
To assess the amount of parent compound in blood, the pooled blood samples were thawed and centrifuged. The remaining blood cells were extracted with acetone/water (50/50, v/v; at pH 2.0), methanol/water (8/2, v/v) and subsequently with acetone. Blood cells of males treated with 100 mg/kg bw/d at the time intervals 2 and 168 h were additionally extracted with methanol and dichloromethane. Organic solvents of the combined extracts were evaporated; protein was precipitated and again extracted. Combined extracts were concentrated and the amount of parent compound was assessed via TLC and a Berthold Automatic TLC-linear Analyser. Mean recoveries were 57.7 ± 10.7 % and 84.3 ± 8.8 % at 3 and 100 mg/kg bw, respectively. The lower recoveries at the low dose may be due to larger counting errors inherent to the low amounts of radioactivity.
ORGANS/TISSUES/ INTESTINAL TRACT/CARCASS: Upon sacrifice, the following organs and tissues were taken: Heart, lung, liver, stomach, spleen, intestinal tract (with contents), adrenal glands, kidney, gonads (ovary/uterus or testicle), muscle, bones (femur), brain, skin (back region), fat, thyroid gland, pancreas and residual carcass. Weights of rats and all specimens mentioned were noted. The radioactivity in organs/tissues was determined after digestion with tissue solubilizer. Radioactivity in the digestive tract and the carcass was assessed after homogenisation and combustion. Bones were combusted directly.
BILE: The bile was collected in 3 h intervals throughout the study from the ductus choleodochus by means of a catheter. The radioactivity was assessed in a LSC.
CAGE WASH: The cages were washed with water and acetone and radioactivity was determined by combustion.
PLASTER AND BANDAGE: Plaster and bandage of operated animals (to collect bile) were extracted with acetone and radioactivity was determined in a LSC.
Total recoveries for both sexes at both dose levels ranged from 91.8 % to 102.3 %.
Remaining samples of organs, bile, blood and plasma were stored at -20 °C for further analysis.

Results and discussion

Preliminary studies:

The aim of the pre-test was to assure that no mortality occurred using the high dose level of the test substance in the formulation.

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The test substance was rapidly and almost completely absorbed in both sexes at both dose levels (Cmax in plasma within 0.5 and 1-2 h after administration at 3 and 100 mg/kg bw, respectively).

Main ADME results 2

Type:

distribution

Results:

Radioactivity in tissues ranged from 0.1% (intestinal tract) to 0.8 % (blood). At 3.0 mg/kg bw the radioactivity in organs/tissues, blood and plasma were below 0.5 µg/g after 168 h. At 100.0 mg/kg bw the residual radioactivity was about 30 times higher.

Main ADME results 3

Type:

excretion

Results:

The test substance was rapidly excreted via feces (76.3 % -79.0 %) and urine (12.4 % -20.7 %) in both sexes at both dose levels. Up to 67.1 % of applied radioactivity was excreted via the bile.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

ABSORPTION/TOXIKOKINETIC
At the low dose level, maximum levels of radioactivity (Cmax) in plasma were reached within 0.5 hour. At the high dose level Cmax-values in the plasma were reached 1-2 h after administration. These results indicate that the test substance was rapidly absorbed. At 33 times higher dose levels, the AUC-values in the plasma were about 30 times higher, indicating that absorption of the test substance was not saturated at the high dose level.

Details on distribution in tissues:

DISTRIBUTION
Low amounts of radioactivity were found in the intestinal tract (0.1 %), organs/tissues (0.2-0.3 %), blood (0.3-0.8 %) and residual carcass (0.3-0.5 %). Negligible amounts were found in the expired air (<0.05 %).
At 168 h, residual radioactivity levels in organs/tissues, blood and plasma were below 0.5 µg/g at 3.0 mg/kg bw. Highest amounts of residual radioactivity were found in the blood (0.219 µg/g and 0.432 µg/g), liver (0.187 µg/g and 0.158 µg/g), kidney (0.075 µg/g and 0.086 µg/g) and spleen (0.062 µg/g and 0.196 µg/g). After repeated (7x) treatment, residual radioactivity levels were only 2-6 times higher after 168 h (except for the Intestinal tract).
At 100.0 mg/kg bw of the 14C-labelled test article, residual radioactivity in organs/tissues were found to be about 30 times higher, ranging from 0.19 µg/g (muscle) to 5.71 µg/g (spleen). Repeated (14x) oral administration of test substance at 3.0 mg/kg bw did not influence the rates and routes of excretion and the residual radioactivity in organs/tissues.

Details on excretion:

EXCRETION
Excretion of radioactivity via the urine represented in male rats, on avergae, 16.8% and 12.4% of the radioactivity administered for the high and low dose level, respectively. In females, the corresponding figures were 20.7% and 17.3%.
The test substance was rapidly excreted in both sexes at both dose levels via the urine. Already 48 h after the administration, on average, 11.8 % -17.6 % of the radioactivity administered were found in the urine. After 168 h, urinary excretion amounted to 12.4 % -20.7 %.
In males and females, the fecal excretion after 168 h accounted for 76.3 % -79.0 % of the radioactivity administered. Together with the cage wash (1.0%-2.4%), total excreted radioactrivity ranged, on average, from 94.8% to 101.1% for both sexes at both dose levels.
Up to 67.1 % of applied radioactivity were excreted via the bile (indicating that radioactivity via the feces had been previously absorbed). Based on urinary, billary and fecal excretion, the test article was almost quantitatively absorbed (89.6 % -99.7 %) at both dose levels in both sexes. Small amounts of radioactivity were found in the intestinal tract (0.1%), organs/tissues (0.2%-0.3%), the residual carcass (0.3%-0.5%) and negligible amounts in the expired air (<0.05%).

The blood/plasma level studies showed that elimination of radioactivity from the plasma was very rapid for both sexes at the low dose level (T1/2: 5.0 and 5.8 h). After single oral administration at the high dose level, the initial plasma half-lives were 31.8 and 34.3 h for males and females, respectively. The shift in Cmax-values and longer half-lives in plasma at the high dose level compared to the low dose level indicated that at this dose level the process of elimination of radioactivity from plasma became saturable.
In the blood, radioactivity levels were initially lower than the corresponding plasma values. However, from 24 h to 168 h after administration, blood levels became 2-11 times higher than the corresponding plasma levels. The parent compound disappeared within 24 h. These results indicate that the metabolites of the test compound were partially associated to blood cells. Accordingly, in both sexes at both dose levels, longer half-lives (66.0-126 h) were found in blood than in plasma (5.0 to 34.3 h).
After repeated (7x) oral administration at the low dose level, half-lives in all organs/tissues of both sexes ranged from 1.9 h (intestinal tract) to 39.5 h (thyroid gland) except for the spleen (T1/2: >168 h). The longer half-life in the spleen may reflect blood levels due to its role in phagocytosis of erythrocytes and cell debris from the blood stream. After single oral administration at the high dose level similar the half-lives in organs/tissues were found, ranging from 0.4 hours (intestinal tract) to 38.8 h (spleen).
A miximal biliary excretion rate was found during the first 6 hours after administration. After low dose administration in males and females, the excreted radioactivity in the 0-6 h bile amounted to 55.1% and 17.6%, respectively. After high dose administration, the corresponding amounts were 14.0% and 3.0%. These results indicate at both dose levels a 3—5 times higher biliary excretion rate in the males as compared to the females. The significant amounts of radioactivity (0.4 % - 1.1 %) still excreted during the last 3 hour-time intervals of each group indicate that for all groups excretion of radioactivity via the biIe was not completed.
Based on the 0-24 hour biliary excretion in males (67.1 %) after low dose administration and the radioactivity excreted in the 0—24 hour feces after single
oral administration (70.1 x), it may be concluded that radioactivity excreted via the feces had been previously absorbed.

For the femaIes at both dose levels delayed excretion may be assumed since high amounts of radioactivity (39.6 % and 35.7 %) were still present in the digestive
tract. Due to the delayed excretion of radioactivity via the biIe at the high dose level in males and at both dose levels in females, it may be assumed that also for
these groups radioactivity excreted via the feces had been previously absorbed. Therefore, in the balance groups the total amount absorbed (urinary and fecal
excretion) after single ora1 administration, ranged from 89.6 % - 99.7 % for both sexes at both dose levels.

As compared to the corresponding groups after single oral administration, no differences were found with respect to urinary and fecal excretion. These results indicate that repeated oral administration of the test substance at the low dose level has no influence on the excretion pattern of 14C-test substance in male and female rats.

Data from the single oral balance studies of both sexes at both target dose levels (3.0 mg/kg and 100 mg/kg) have shown that the test substance was excreted via the urine in considerabIe amounts (12.4 % - 20.7 %). However, the major route of excretion was via the feces (76.3 % - 79.0 %).

Toxicokinetic parameters

Toxicokinetic parameters 1

Toxicokinetic parameters:

half-life 1st: 5.0 and 5.8 h from plasma

Remarks:

low dose

Toxicokinetic parameters 2

Toxicokinetic parameters:

half-life 2nd: 31.8 and 34.3 h from plasma

Remarks:

high dose

Toxicokinetic parameters 3

Toxicokinetic parameters:

Cmax: 0.5 h

Remarks:

low dose

Toxicokinetic parameters 4

Toxicokinetic parameters:

Cmax: 1-2 h

Remarks:

high dose

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

Taking into account the rapid dissappearance of the parent compound within 24 h, these results indicate that the metabolites of the test substance were partially associated to blood cells. In addition, results indicate that 14C—test substance and/or its metabolites will neither be accumulated nor retained by the rat after repeated oral administration.