2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Remarks:

absorption, distribution, excretion and metabolism of the parent compound in the rat following a single administration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 Sep 2004 - 24 Jan 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted Jul 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: PMRA Ref.: DACO 4.5.9 Metabolism/Toxicokinetics in Mammals (Lab. Animal)

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF New Test Guidelines for Supporting Registration of Chemical Pesticides, 12 Nousan 8147

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Cas Number:

473278-76-1

Molecular formula:

C20 H23 Cl O7 S

IUPAC Name:

2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: >99%
- Specific activity: 4.96 MBq/mg = 2.976 x 10 E5 dpm/μg = 133.92 μCi/mg = 59.31 Ci/mol
- Locations of the label: Phenyl-UL-14C

Radiolabelled reference items M 2 – M 7 originated from rat urine sample (male, high dose) of a previous study [M-265729-01, 2006] and the radiolabelled reference item M 1 from a rat urine sample (male, high dose). All components were purified to near homogeneity within this study and identified by spectroscopic methods (LC-MS and NMR). They were stored in laboratory deep freezers at about -20°C or refrigerators at about +4°C.

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Wistar Hsd/Cpb: WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan & Winkelmann GmbH, Borchen, Germany.
- Age at study initiation: 8 weeks (males), 9 - 11 weeks (females).
- Weight at study initiation: Males: 186 - 209 g; females: 178 - 198 g.
- Housing: After administration of the radiolabelled test compound, the rats were kept individually in Makrolon® metabolism cages. With these cages, an almost quantitative and separate collection of urine and faeces was possible.
- Diet: Rat/mice maintenance long life diet (no. 3883.0.15, supplied by Provimi Kliba AG,Kaiseraugst, Switzerland), ca. 16 g per animal and day, ad libitum. the animals were fasted from approximately 16 h prior to dosing until approximately 6 h after dosing.
- Water: Tap water from the local mains supply, ad libitum.
- Acclimation period: Approximately 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Approximately 25
- Humidity (%): 50 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth®

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The radiolabelled test substance was received in solid form, and stock solution was prepared by dissolving it in acetonitrile. For the preparation of the administration suspensions an amount of 5.427 mL (= 2.16 mg test compound) of the radioactive stock solution was pipetted into a glass flask and concentrated under a gentle stream of nitrogen. The nearly dry residue was suspended in 10.8 mL 0.5% aqueous Tragacanth® and the suspension was stirred overnight at ca. +4°C and during the whole administration process at room temperature on a magnetic stirrer. The administration suspensions were prepared one day before dosing. The target dose was 2 and 200 mg/kg bw, in a volume of 2 mL.

Duration and frequency of treatment / exposure:

Single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 other: mg/kg bw (nominal)

Remarks:

Actual dose: 2.03 mg/kg bw (males); 2.29 mg/kg bw(females)

Doses / concentrations 2

Dose / conc.:

200 other: mg/kg bw (nominal)

Remarks:

Actual dose: 199.49 mg/kg bw (males); 209.88 mg/kg bw (females)

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood (erythrocytes, plasma); gastrointestinal tract, organs/tissues (spleen, liver, kidney,perirenal fat, adrenal gland, ovaries, uterus, testis, skeletal muscle, femur, heart, lung, brain, thyroid gland, harderian gland, skin, eye, carcass).
- Time and frequency of sampling:
Blood: Collected at 0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 24, 32, 48, 46, 72 h post-administration. Blood samples were collected separately for each animal by pressing a capillary coated with heparin in a small cut in the tail vein. The wound was closed with adhesive tape. The capillaries were centrifuged at ca. 12,000 g for 10 minutes using a hematocrit centrifuge to separate the plasma from the formed blood constituents (mainly erythrocytes). After centrifugation, the capillary was broken at the border between plasma and formed constituents and the plasma (ca. 10 - 80 mg) was pressed onto a small metal dish for weighing. The dish was then placed into a scintillation vial for radioactivity measurement.
Urine: Collected at 4, 8, 24, 48, 72 h post-administration; separately for each animal in a cryogenic trap cooled with dry ice. The funnels for urine collection were rinsed with demineralised water at the end of each sampling period. The rinsing solutions were drained into the same vial as the corresponding urine fraction.
Faeces: Collected at 24, 48, 72 h post-administration; separately for each animal in a cryogenic trap before they were lyophilised (freeze-dried), weighed, and homogenised.
Tissues: The retained tissues were weighed immediately after the dissection and again following lyophilisation. Finally, they were homogenised before aliquots were taken for the determination of radioactivity by combustion/LSC. For the small organs and tissues (e.g. renal fat, uterus, harderian gland, pancreas, adrenal glands, thyroid, ovaries and eye), only the wet weight was determined before they were solubilised using BTS 450® (Beckman Tissue Solubiliser).

- From how many animals: Samples of urine and faeces were pooled per sex and time period. Urine: low-dose males: 0 – 4 h, 4 – 8 h, 8 – 24 h; low-dose females: 0-24 h; high-dose males: 0-24 h; high-dose females: 0-24 h. Faeces: low-dose males: 0 – 24 h, 24 – 48 h; low-dose females: 0-48 h; high-dose males: 0-48 h; high-dose females: 0-48 h.
- Method type(s) for identification: Liquid scintillation counting (blood, urine); combustion/LSC (radioactivity in faeces, tissues and organs).

- Other:
The total radioactivity in the test substance and metabolites was determined in the excreta (urine and faeces) as well as in blood, gastrointestinal tract, organs/tissues. The metabolism was investigated by radio-HPLC and spectroscopic methods in selected urine samples as well as in faeces extracts.
The software TOPFIT version 2.0 (Heinzel et al., 1993) was used to calculate the toxicokinetic parameters by plasma concentration-time curve analysis for the mean equivalent concentration-values of each test. A standard 4-compartment disposition model was applied for curve fitting computation. Compartments are defined as physical locations in the body that can be represented with certain simplifications during modelling. Compartment models attempt to describe the following processes mathematically: absorption of the administered drug, entry into the systemic circulation, distribution to organs or tissues where metabolism can occur, and subsequent excretion. The correlation coefficients were acceptable for all tests (0.96 – 0.99).
Sacrifice: 72 h after substance administration, the animals were anaesthetised using Pentobarbital-Na (Narcoren®, supplied by Merial GmbH, Hallbergmoos, Germany) and sacrificed by transection of the cervical blood vessels.
Test #: 1 = male, low-dose; 2 = female, low-dose; 3 = male, high-dose; 4 = female, high-dose.


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces, blood (erythrocytes, plasma); gastrointestinal tract, organs/tissues (spleen, liver, kidney,perirenal fat, adrenal gland, ovaries, uterus, testis, skeletal muscle, femur, heart, lung, brain, thyroid gland, harderian gland, skin, eye, carcass).
- Time and frequency of sampling:
Urine: Collected at 0 – 4 h, 4 – 8 h, 8 – 24 h and 24 – 48 h separately for each animal in a cryogenic trap cooled with dry ice. The funnels for urine collection were rinsed with demineralised water at the end of each sampling period. The rinsing solutions were drained into the same vial as the corresponding urine fraction.
Faeces: Collected at 0 – 24 h and 24 – 48 h separately for each animal in a cryogenic trap before they were lyophilised (freeze-dried), weighed, and homogenised.
Blood: After transsection of the cervical blood vessels, the oozed out blood was collected into test tubes coated with heparin that was separated afterwards into plasma and erythrocytes by centrifugation.
Tissues: The retained tissues were weighed immediately after the dissection and again following lyophilisation. Finally, they were homogenised before aliquots were taken for the determination of radioactivity by combustion/LSC. For the small organs and tissues (e.g. renal fat, uterus, harderian gland, pancreas, adrenal glands, thyroid, ovaries and eye), only the wet weight was determined before they were solubilised using BTS 450® (Beckman Tissue Solubiliser).
- From how many animals: Samples of urine and faeces were pooled per sex and time period. Urine: low-dose males: 0 – 4 h, 4 – 8 h, 8 – 24 h; low-dose females: 0-24 h; high-dose males: 0-24 h; high-dose females: 0-24 h. Faeces: low-dose males: 0 – 24 h, 24 – 48 h; low-dose females: 0-48 h; high-dose males: 0-48 h; high-dose females: 0-48 h.
- Method type(s) for identification: Liquid scintillation counting (blood, urine); combustion/LSC (radioactivity in faeces, tissues/organs); HPLC (purity check of the stock and administration solutions of the test substance; metabolic profiling and quantitative analysis of urine, bile, faeces); LC-MS/MS to confirm identity of substance and metabolites (urine).

- Limits of detection and quantification: For all samples, the limit of detection (LOD) was established at ca. 20 dpm measured per aliquot after correction for the background radioactivity. Only in very rare cases, lower values than 20 dpm were accepted. The limit of quantitation (LOQ) for each individual measurement was established as 2 times of the background radioactivity (dpm) of each instrument/method. The respective value was printed out on the original raw data sheet. This background counting rate was in a range between 10 - 35 cpm (approximately equal to 10 – 35 dpm) and it was automatically subtracted from the measuring results. A quench and counting efficiency correction for transformation of gross counts (cpm) into net counts (dpm) was automatically performed by the instruments. Samples with individually measured values below two times of the background radioactivity and for which the average counting efficiency was lower than 50% and the percentage error greater than 10% were not quantified.

Statistics:

All calculations (e.g. radioactivity in the administration suspensions, balances of radioactivity during sample preparation, balances of extraction) were performed mainly using Microsoft Excel® software. The fundamental calculations were based on the LSC results, expressed in disintegrations per minute (dpm-values). The dpm-value of each aliquot shown in the tables or appendices was generally the arithmetic mean - rounded to an integer number - from at least 2 measurements (liquid samples) or at least 2 combustion values (solid samples). The only exception from this procedure was the measurement of the plasma micro samples for which the dpm-value of only one aliquot was determined.

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

low-dose males: 94.35%; low-dose females: 94.35%; high-dose males: 97.46%; high-dose females: 87.37%

Main ADME results 2

Type:

distribution

Results:

5.622% (low-dose males), 6.259% (low-dose females), 0.131% (high-dose males) and 0.146% (high-dose females) of the administered dose, respectively, was recovered from the blood, organs and tissues (not including gastrointestinal tract)

Main ADME results 3

Type:

metabolism

Results:

In males > 60% of the parent compound was metabolised, in females > 30% was metabolised. The tetrahydrofurane ring of the molecule was the preferred site for metabolism. Parent compound-hydroxy-pentanoic acid (M 4) was the dominant metabolite.

Main ADME results 4

Type:

excretion

Results:

Total elimination: 89.65% for low-dose male rats, 88.05% for low-dose female rats, 97.32% for high-dose male rats, 87.20% high-dose female rats.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The oral absorption rate of the test substance was calculated from the recoveries in the urine, bile and organs/tissues: 94.35% in the low-dose males, 94.35% in the low-dose females, 97.46% in the high-dose males, and 87.37% in the high-dose females, respectively of the administered dose were recovered from measurement of the total radioactivity in urine and faeces as well as in organs and tissues at sacrifice. The relatively lower rate observed in high-dose females may possibly be attributed to the administration of a marginal lower amount of the highly viscous suspension as calculated from calibration of the administration suspension.
The test substance was very rapidly absorbed from the gastrointestinal tract of male and female rats in all treatment groups. The absorption started immediately after oral dosing as shown by the plasma curves and the values calculated for the absorption half-lives (see Table 1 and 2 under "Any other information on results incl. tables"). No significant differences were observed between the low- and high-dose groups, or between males and females. Although no exact value for the absorption rate could be taken from these figures it can be assumed that most of the administered dose was absorbed and systemically available.

Details on distribution in tissues:

The distribution of the test substance from the central compartment to the different organs and tissues was followed by measuring the concentration of the total radioactivity in plasma (see 'Toxicokinetic parameters' and Table 2 under "Any other information on results incl. tables"). The maximum plasma concentration (Cmax) value was significantly higher for low-dose males (3.4 μg/g) than for low-dose females (1.9 μg/g). The maximum absorption time (tmax) was slightly higher for the low-dose males (0.3 h) compared with the low-dose females (0.1 h). This was followed by a fast initial elimination phase (t1/2e(1)) of 1.1 h (low-dose males) and 0.3 h (low-dose females), a slower intermediate elimination phase (t1/2e(2)) of 2.4 h (low-dose males) and 3.0 h (low-dose females) and a moderate terminal elimination phase (t1/2e(3)) of 17.8 h (low-dose males) and 27.3 h (low-dose females). The area under the curves (AUC(0-∞)) indicated a slightly higher systemic exposure for low-dose males (4.0 μg/g x h) than for low-dose females (2.7 μg/g x h). In low-dose males, significantly lower values were calculated for the elimination rate constant (k1e = 4.7 h) and the mean residence time (MRT = 2.0 h) compared with the corresponding values in low-dose females (k1e = 48.5 h; MRT = 9.1 h).

Similar Cmax values were noted for high-dose males (277 μg/g) and high-dose females (284 μg/g). Compared with the low dose tests, these plasma concentrations were nearly proportional to the dose ratio. This indicated that the absorption process was not (over)saturated at the high-dose level. The maximum absorption time (tmax) was higher in the high-dose animals, compared with the low-dose animals, and relatively similar in males (1.0 h) and females (0.8 h). This was followed by a fast initial elimination phase (t1/2e(1)) of 0.1 h (high-dose males) and 0.5 h (high-dose females), a slower intermediate elimination phase (t1/2e(2)) of 2.0 h (high-dose males) and 1.4 h (high-dose females) and a moderate terminal elimination phase (t1/2e(3)) of 16.0 h (high-dose males) and 12.3 h (high-dose females). The area under the curves (AUC(0-∞)) indicated a slightly higher systemic exposure for high-dose males (1250 μg/g x h) than for high-dose females (933 μg/g x h). In high-dose males, a higher value was calculated for the elimination rate constant (k1e = 30.5 h) and a slightly lower for the mean residence time (MRT = 4.0 h), compared with the corresponding values in high-dose females (k1e = 12.8 h; MRT = 4.2 h).

The comparison of the absorption phases of the kinetic curves between the low- and high-dose groups showed a broader maximum for the high-dose animals. The maximum concentration (Cmax) was reached later at the high-dose level than at the low-dose level, and the following initial elimination phase was definitely longer in the high-dose than in the low-dose level. The curves at the end of the terminal elimination phase were comparable between the sexes. The AUC-values for males and females of the low dose and high dose groups indicated that the parent compound and/or its metabolites showed a disproportionately higher systemic exposure at the high dose level (i.e. >300-fold), which was probably due to an apparent saturation of the initial elimination/biotransformation processes.

The radiolabelled residues in the organs and tissues of the animals were determined at sacrifice, 72 h after the oral administration. Negligible amounts of radioactivity was found in the skin (0.01 - 0.02%) and gastrointestinal tract (0.01 - 0.04%). No significant sex-related differences were observed for the residues in the organs and tissues. In the low-dose animals, 5.6 and 6.3% of the administered dose was detected in the organs of male and female rats, respectively. In the high-dose animals, low percentages (0.1%) were found in the organs of both sexes. The highest equivalent concentrations were detected in the liver (2.1 - 5.4 ppm) and kidney (0.6 - 1.9 ppm), which are the organs responsible for the degradation and excretion of the test substance. These values were not correlated with the administered dose between the low- and high-dose groups. It is likely that tissue binding sites were more or less saturated with test item related radioactivity already at the low-dose level. The concentrations in the other organs and tissues (except gastrointestinal tract) were low (0.0005 - 0.0067 ppm in the low-dose groups and 0.04 - 0.65 ppm in the high-dose groups). From the renal and faecal excretion and from the elimination kinetics of total radioactivity from plasma it was concluded that the small amounts of residual radioactivity in the other organs and tissues are subject to further elimination.

Details on excretion:

The excretion was fast: the urinary excretion was almost completed 24 h after administration and the faecal excrection was almost complete 48 h after dosing.
Significant sex differences were observed. In the low-dose male rats, 8.78% of the dose was excreted with the urine and 80.87% with the faeces. In the low-dose female rats, relatively more radioactivity was eliminated via the urine (31.44%) and less via the faeces (56.61%), compared with the low-dose males. The renal to faecal ratio was 0.11 in low-dose males and 0.56 in low-dose females.
In the high-dose male rats, 15.90% of the dose was excreted with the urine and 81.42% with the faeces. In the high-dose female rats, more radioactivity was eliminated via the urine (56.00%) than via the faeces (31.20%), in contrast with the low-dose female group and both males groups. The renal to faecal ratio was 0.20 in high-dose males and 1.79 in high-dose females. The higher renal excretion in these tests is possibly due to preferred elimination of the unchanged parent compound combined with biliary excretion of metabolites.
The HPLC-profiles of radioactivity in the urine and the faeces were similar between all the groups. The urine HPLC-profile of the 0 – 24 h collection period of high-dose males (test 3) and the faeces HPLC-profiles of the 0 – 24 h and 24 – 48 h collection periods of low-dose males (test 1) were highly comparable.

Toxicokinetic parameters

Toxicokinetic parameters 1

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 1.07

Toxicokinetic parameters 2

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 0.30

Toxicokinetic parameters 3

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 0.07

Toxicokinetic parameters 4

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 0.45

Toxicokinetic parameters 5

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 2.37

Toxicokinetic parameters 6

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 2nd: 2.95

Toxicokinetic parameters 7

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 2nd: 1.97

Toxicokinetic parameters 8

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 2nd: 1.41

Toxicokinetic parameters 9

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 3rd: 17.80

Toxicokinetic parameters 10

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 3rd: 27.30

Toxicokinetic parameters 11

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 3rd: 16.00

Toxicokinetic parameters 12

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 3rd: 12.30

Toxicokinetic parameters 13

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 3.99

Toxicokinetic parameters 14

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 2.71

Toxicokinetic parameters 15

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 1250

Toxicokinetic parameters 16

Key result

Test no.:

#4

Toxicokinetic parameters:

AUC: 933

Toxicokinetic parameters 17

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 3.43

Toxicokinetic parameters 18

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: 1.91

Toxicokinetic parameters 19

Key result

Test no.:

#3

Toxicokinetic parameters:

Cmax: 277.0

Toxicokinetic parameters 20

Key result

Test no.:

#4

Toxicokinetic parameters:

Cmax: 284.0

Toxicokinetic parameters 21

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 0.31

Toxicokinetic parameters 22

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 0.13

Toxicokinetic parameters 23

Key result

Test no.:

#3

Toxicokinetic parameters:

Tmax: 1.0

Toxicokinetic parameters 24

Key result

Test no.:

#4

Toxicokinetic parameters:

Tmax: 0.8

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The identification rate of parent compound and metabolites was high and amounted in total to 82.2% of the administered dose in low-dose males (test 1), 82.3% in low-dose females (test 2), 91.2% in high-dose males (test 3) and 83.1% in high-dose females (test 4) (see Table 3 under "Any other information results including tables"). Unknown metabolites accounted in total for < 1.01% of the dose in the urine samples and < 5.37% in the faeces extracts. No single unknown metabolite was higher than 0.5% of the dose in the urine and 1.3% in the faeces.
The parent compound was of minor importance in the low-dose male rats with only 0.07% of the dose in the urine and 3.81% in the faeces. In the low-dose female rats, 20.29% of the parent compound was excreted unchanged with urine and 2.21% with faeces. Comparable proportions for the parent compound were recovered from the excreta of the respective high-dose animals. In high-dose male rats, 9.69% and 5.37% of the administered parent compound was recovered from the urine and faeces, respectively. Significantly higher values (51.91% in the urine and 6.16% in the faeces) were measured for high-dose female rats. Significant sex differences were seen for the amount of metabolites detected in the respective samples. Quantitative higher amounts of metabolites were generally found in the respective faeces extracts. The extent of degradation of the parent compound to the different metabolites was greater in males than in females. The tetrahydrofurane ring of the molecule was the preferred site for degradation. No metabolic changes were detected at the phenyl ring and only minor ones at the cyclohexyl ring. Two major metabolites, hydroxy-pentanoic acid (M 4), and 5-oxo-THF (M 7), and to a less extent dihydroxy-pentyl (M 3) as well as the metabolite region of M 5 (M 5A: oxo-pentanoic acid, M 5B1: 2-hydroxy-THF and M 5B2: hydroxy-oxo-pentyl) were identified. Of the above-mentioned, the major metabolite was hydroxy-pentanoic acid (M 4), accounting for 66.50% and 43.57% in the excreta of male and female rats of the low dose tests as well as 45.96% and 16.73% in the excreta of the high-dose males and females, respectively. Significantly lower values were measured for the other metabolites. 5-oxo-THF (M 7), dihydroxy-pentyl (M 3) and oxo-pentanoic acid (M 5A) were ≤ 9.27% of the dose in excreta of the low-dose females and 11.74% in the high-dose males, respectively. The hydroxylated metabolite 4-hydroxy-cyclohexanedione (M 6) was only found in the high-dose groups and accounted for 5.37% and 2.54% of the dose in excreta of male and female rats, respectively. The cleavage of the tetrahydrofurane ring leading to the urinary metabolites benzylic alcohol (M 2) and hydroxy-benzylic alcohol (M 1) was a relatively insignificant reaction, since the values for these metabolites did not exceeded 0.8% of the administered dose.
The proposed biotransformation pathway of the parent compound is attached under "Attached background material". The first metabolic reaction was an oxidative and obviously important step at the tetrahydrofurane ring of the molecule by which parent compound-oxo-THF was formed. Hydrolytic cleavage of the lactone ring led to parent compound-hydroxy-pentanoic acid which was the dominant metabolite in all treatment groups. On a lower quantitative level, further changes at the ring-opened structure occurred. Hydroxylation at the the cyclohexyl- and tetrahydrofurane-ring of the test item as well as cleavage of the tetrahydrofurane ring were of minor importance.

Any other information on results incl. tables

Table 1: Balance of radioactivity in excreta, and organs and tissues of male and female rats following a single oral administration of 2 and 200 mg/kg bw

Treatment group	2 mg/kg bw	2 mg/kg bw	200 mg/kg bw	200 mg/kg bw
Sex	Male	Female	Male	Female
Radioactivity (%) of administered dose (mean values of 5 animals/sex)
Urine	8.78	31.44	15.90	56.00
Faeces	80.87	56.61	81.42	31.20
Total excreted	89.65	88.05	97.32	87.20
Renal/faecal ratio	0.11	0.56	0.20	1.79
Skin	0.017	0.005	0.009	0.016
Sum organs/tissues	5.605	6.254	0.121	0.131
Body without gastrointestinal tract	5.622	6.259	0.131	0.146
Gastrointestinal tract	0.033	0.044	0.009	0.024
Total in body	5.654	6.303	0.140	0.171
Balance	95.30	95.35	97.46	87.37

 

Table 2: Distribution and plasma kinetics

Treatment group	2 mg/kg bw	2 mg/kg bw	200 mg/kg bw	200 mg/kg bw
Sex	Male	Female	Male	Female
t1/2 a[h]	0.13	0.004	0.003	0.006
k1e[1/h]	4.74	48.50	30.5	12.8
CL/f [mL/min/kg bw]	8.36	12.30	2.66	3.57
CLR[mL/min/kg bw]	0.73	3.87	0.42	2.00
MRT [h]	1.95	9.11	4.02	4.16
MRTabs[h]	0.60	0.53	0.99	0.79
MRTdisp[h]	1.35	8.59	3.04	3.36

t_{1/2 a}=half-life of the absorption

k_1e=Total (renal, biliary, metabolic, …,) elimination rate constant from the central compartment

CL/f =Total clearance of radioactivity from plasma assuming a complete absorption process

CL_R=Renal clearance of radioactivity

MRT =Mean residence time describing the sojourn (elimination and/or metabolism) of the drug or total radioactivity in the body

 

 

Table 3: Quantitative evaluation of parent compound and metabolites in the urine and faeces of male and female rats following a single oral administration of 2 and 200 mg/kg bw

Treatment group	2 mg/kg bw, male			2 mg/kg bw, female			200 mg/kg bw, male			200 mg/kg bw, female
Sample	Urine	Faeces	Total	Urine	Faeces	Total	Urine	Faeces	Total	Urine	Faeces	Total
Sample period [h after admin.]	0 – 24 h	0 – 48 h		0 – 24 h	0 – 48 h		0 – 24 h	0 – 48 h		0 – 24 h	0 – 48 h
	% of dose administered			% of dose administered			% of dose administered			% of dose administered
Residue in analysed samples	8.70	80.58	89.28	30.64	56.31	86.95	15.72	81.12	96.84	54.60	30.53	85.13
Peak ID
U 1	-	0.69	0.69	-	0.29	0.29	-	0.53	0.53	-	-	-
M 1	0.03	-	0.03	-	-	-	0.13	-	0.13	-	-	-
U 2	0.01	0.25	0.26	-	-	-	0.07	0.65	0.72	-	-	-
U 3	0.14	1.28	1.42	0.14	1.06	1.20	-	0.68	0.68	-	0.21	0.21
U 4	-	-	-	-	-	-	0.08	0.66	0.73	-	0.20	0.20
U 5	0.06	1.01	1.07	-	0.52	0.52	-	0.61	0.61	-	-	-
M 2	0.13	-	0.13	0.18	-	0.18	0.80	-	0.80	0.16	-	0.16
U 6	0.04	0.46	0.51	0.05	-	0.05	-	-	-	-	0.13	0.13
M 3	0.73	2.33	3.05	1.12	3.70	4.82	0.14	5.64	5.79	0.20	2.26	2.45
M 4	6.05	60.45	66.50	3.50	40.07	43.57	2.65	43.31	45.96	0.72	16.01	16.73
U 7	0.06	0.67	0.73	-	0.44	0.44	-	0.27	0.27	-	-	-
U 8	0.12	0.68	0.80	0.31	0.47	0.78	-	0.58	0.58	0.09	-	0.09
M 5A	0.27	2.90	3.18	0.14	1.29	1.43	0.65	11.09	11.74	0.05	0.87	0.92
M 5B1/M 5B2	0.16	0.33	0.49	0.42	0.07	0.49	0.67	3.10	3.77	0.15	0.14	0.29
U 9	0.03	-	0.03	-	-	-	0.07	-	0.07	-	-	-
U 10	0.02	-	0.02	0.23	-	0.23	-	-	-	0.10	-	0.10
M 6	-	-	-	-	-	-	0.50	4.87	5.37	0.36	2.18	2.54
M 7	0.77	4.11	4.89	3.98	5.29	9.27	0.26	2.32	2.58	0.38	1.57	1.95
U 11	-	0.32	0.32	0.15	-	0.15	-	0.47	0.47	0.25	0.36	0.60
M 8	0.07	3.81	3.88	20.29	2.21	22.50	9.69	5.37	15.06	51.91	6.16	58.06
U 12	-	-	-	0.14	-	0.14	-	-	-	0.24	-	0.24
Sum identified	8.22	73.94	82.16	29.63	52.63	82.26	15.50	75.70	91.20	53.92	29.18	83.10
Renal/faecal ratio of identified compounds	9.0			1.8			4.9			0.5
Sum unknown	0.48	5.37	5.85	1.01	2.79	3.80	0.22	4.45	4.67	0.68	0.90	1.58
Subtotal	8.70	79.31	88.01	30.64	55.41	86.05	15.72	80.15	95.87	54.60	30.08	84.68
Solids	-	1.25	1.25	-	0.88	0.88	-	0.95	0.95	-	0.44	0.44
Not analysed	-	0.02	0.02	-	0.02	0.02	-	0.02	0.02	-	0.01	1.01
Total	8.70	80.58	89.28	30.64	56.31	86.95	15.72	81.12	96.84	54.60	30.53	85.13
Not analysed (urine 24 – 72 h)			0.08			0.80			0.18			1.40
Not analysed (faeces 48 – 72 h)			0.29			0.30			0.30			0.67
Sum total			89.65			88.05			97.32			87.20