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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Remarks:

distribution and excretion of the parent compound in the rat following a single oral administration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Mar 2003 - 23 Jan 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 04 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

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: 9 weeks (males), 11 weeks (females).
- Weight at study initiation: Males: 199 - 219 g; females: 190 - 202 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): 20 - 24
- Humidity (%): 32 - 61
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

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 in two portions, and stock solutions were prepared by dissolving the test substance in acetonitrile. For the preparation of each of the administration suspensions a known amount 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 a known amount of 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 3 mg/kg bw, in a volume of 2 mL.

The dosing solutions for the non-radiolabelled test substance were prepared according to the same method as described above for the radiolabelled test substance. The target dose was 3 mg/kg bw, in a volume of 2 mL.

Duration and frequency of treatment / exposure:

Single treatment

Dose / conc.:

3 other: mg/kg bw (nominal)

Remarks:

Actual dose: 2.71 mg/kg bw (males); 3.15 mg/kg bw(females)

No. of animals per sex per dose / concentration:

8 (treatment group, radiolabelled test substance)
1 (control group, non-radiolabelled test substance)

Control animals:

yes

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood; organs/tissues (spleen, liver, renal cortex, renal medulla, perirenal fat, skeletal muscle, myocardium, lung, pancreas, bone marrow, testes (males), uterus (females), ovary (females), brain, spinal cord, pituitary gland, pineal body, adrenal gland, thymus, thyroid gland, salivary gland, nasal mucosa, skin, eye)
- Time and frequency of sampling: The animals (1/sex/time period) were sacrificed using carbon dioxide 1, 4, 8, 24, 48, 72, 120 and 168 h post-administration. The control animals were sacrificed 4 h after dosing. Sample collection schedule for urine and faeces per animal are given in Table 1 under "Any other information on materials and methods incl. tables".
Urine: Collected 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 separately for each animal in a cryogenic trap before they were lyophilised (freeze-dried), weighed, and homogenised.
Expired air: carbon dioxide and other volatiles from expired air were collected from 4 male animals and 4 female animals. The respective metabolism cages were attached to a high velocity air pump and ventilated with ca. 2 L of air per minute and cage. The out coming air was passed through a trapping system of two gas-washing bottles each containing about 150 – 200 mL of a 1:1-mixture of ethanolamine/ethanol. At sampling, the exact volume was determined, from which an aliquot was taken for the determination of radioactivity.
- Method type(s) for identification:
Quantitative whole body autoradiography (QWBA) using the radioluminography (RLG) technique was used to quantify the amount of radiolabelled substance in tissues and organs. This method allows the visualisation of selective enrichments of radioactivity, particularly in certain parts of organs or tissues, which are difficult or impossible to sample during the sectioning of the animal. After sacrifice, the animals were fixed in a stretched position using a metal template and immediately frozen at ca. -70°C. After removal of the template, the animal body together with the series of blood standards was embedded in a slurry of carboxymethylcellulose (7 to 8 %) on the platform of the microtome. The sections (4-5/animal) were cut at a thickness of 50 μm, attached to adhesive tape, and freeze-dried overnight in the cooling cabinet of the microtome. Two imaging plates - designated as sections A and B - were exposed to the sections at time ranges from 48 to 120 hours. The exposed imaging plates were processed. In order to correct for possible chemo-graphic effects, the non-radiolabelled test item was orally administered at the same dose to one male (no. 658) and one female control animal (no. 667). The control animals were sacrificed after 4 hours. The sections were exposed under identical conditions, using the longest exposure time which had been chosen for rat sections of animals treated with the radiolabelled compound. The digital images of the radioluminograms allowed for the assessment of the distribution of radioactivity concentrations in different organs and tissues.
Quantification of Whole Body Autoradiography (Radioluminography): The radioactivity was quantified using the software "Tina®" (raytest, version 2.10g). Defined areas were set and integrated in each organ or tissue or partial structure thereof. After background subtraction, a value of the photostimulated luminescence (PSL) per mm² was obtained, which is proportional to the equivalent concentration of the radioactivity in that particular tissue. Two series of 7 calibration standards were prepared by spiking bovine control blood with different concentrations of 14C-radiolabelled Enrofloxacin®. The radioactivity of each blood calibration standard was determined after combustion/LSC. The mean values of each standard were used to establish a calibration graph for the correlation of (PSL – Bkg)/ mm² to the activity in dpm/g tissue. After integration and background (Bkg) correction, the relationship between (PSL –Bkg)/mm² and dpm/g tissue was established by linear regression analysis. The obtained regression factors were used to calculate the concentration of the radioactivity in dpm/g in the lyophilised rat sections.To express these values as equivalent concentrations, the radioactivity concentrations given in dpm/g were divided by the specific radioactivity of the test substance given in dpm/μg.The self-absorption of the various tissues depends upon several factors like the consistency of the biological material (e.g. bone, muscle, fat) and its shrinking behaviour during lyophilisation. A correction for this self-absorption was made based on earlier Investigations.
The total radioactivity was determined in the excreta (urine and faeces) and in the exhaled 14CO2 by LSC.

- Limits of detection and quantification:
Urine and faeces: For all samples, the limit of detection (LOD) was established at ca. 20 dpm measured per aliquot after correction for the background radioactivity. The only exception was the measurement of radioactivity in the expired air samples for which all reasonable dpm values were quantified. The limit of quantification (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 12 – 30 cpm (approximately equal to 12 – 30 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 and labelled as < LOD in the respective tables.
QWBA: For the measurement of radioactivity in the organs and tissues with radioluminography, the limit of detection (LOD) in dpm/g was established as the 2-fold background radioactivity (PSL/mm2) for each imaging plate. As the regression factor is calculated from background corrected PSL values, the LOD in dpm/g corresponds to the 2-fold background radioactivity. The LOD for the organs and tissues of each individual animal was the average calculated from the LOD values of the two imaging plates prepared. The limit of quantification (LOQ) was set either at the 2-fold of the respective LOD-value or the lowest value of the calibration standards which was included in the calibration graph, whichever was higher. The LOQ for the organs and tissues of each individual animal was the average calculated from the LOQ values of the two imaging plates prepared.
Other: HPLC-analysis was used for purity checking of the test item in the stock solutions and administration suspensions.

Type:

distribution

Results:

The test substance was rapidly and extensively distributed in all organs and tissues, with the highest concentrations in the liver and kidney.

Type:

excretion

Results:

> 90% of the test substance was excreted within approximately 48 hours. In male rats ca. 90% faecal excretion and 10% renal excretion; in female rats ca. 60% faecal excretion and 40% renal excretion.

Details on absorption:

The test substance was readily absorbed from the gastrointestinal tract (see Table 4-5 under 'Any other information on results incl. tables' for total excretion rate).

Details on distribution in tissues:

Qualitative distribution:
In general, the more intense the blackening of the radioluminograms, the higher is the concentration of the radioactivity in the organs or tissues. One hour after oral administration, the radioactivity was distributed among almost all organs and tissues. The most intense blackening was found in the stomach, the small intestine, the urinary bladder, the liver, and the kidney. Less blackening was found in the blood, heart, and lung (males) and blood, brown (subcutaneous) fat, adrenal gland, uterus, ovary, thyroid, heart, and lung (females), respectively, followed by the other organs and tissues with decreasing intensity of blackening. Little blackening was found in the nasal mucosa, brain, spinal cord, and vitreal body of the eye. These results demonstrate that the test substance was readily absorbed. 4 hours after dosing the radioactivity in all peripheral tissues has decreased considerably with only weak blackening to be seen. Significant blackening can still be detected in the blood, heart, lung, spleen and testes (males), and blood, heart, lung, spleen, adrenal gland, thyroid and ovary (females), respectively. The highest concentrations of radioactivity are present in the excretory organs kidney, liver and in the gastro-intestinal tract for males and females. 8 hours after oral administration, the result was similar to the previous one. The most intense blackening was found in the contents of the large and small intestine as well as in the liver and kidney. 24 hours after administration, the extent and intensity of blackening in most organs and tissues had further decreased in the males. High blackening was still observed in the contents of the large intestine, the kidney and the liver. In the females, 24 hours after administration, no blackening above the background could be observed for the tissues and organs, except kidney, liver and gastrointestinal tract. Weak blackening could be seen in blood, adrenal gland, heart, and lung. In the radioluminograms of the rats sacrificed 48 hours, 72 hours, 120 hours, and 168 hours after oral administration, no blackening above the background could be observed for all organs in tissues except kidney, liver, and the gastro-intestinal tract. These results demonstrate the fast depletion of test substance-related radioactivity from all peripheral compartments of the rat. High blackening at a constant level between 48 and 168 hours was observed for liver and kidney indicating a retention and very slow elimination from these organs.

Quantitative distribution: Among the quantitatively analysed organs, tissues, and fluids, the highest equivalent concentrations were observed in the liver, kidney and blood (see Table 4-5 under 'Any other information on results incl. tables'). Moderate peak concentrations were found in the lung and myocardium, brown fat, skin, salivary gland, thyroid, adrenal gland and the reproduction organs. Lower concentrations were reached in all other organs and tissues. The lowest peak concentrations were found in the spinal cord, the brain, and the eye. From peak values, a continuous decrease of radioactivity concentrations by several orders of magnitude, below the limit of quantification was observed for most organs and tissues within 48 hours in male rats and within 24 hours in female rats. High concentrations of radioactivity persisted in kidney and liver of both male and female rats for the whole test period of 7 days indicating significant retention and very slow elimination from these organs.

Details on excretion:

The test substance was excreted rapidly; within approximately 48 hours, as indicated by the time course of the total cumulative excretion of urine, faeces and expired air. In male rats primarily faecal excretion was observed (ca. 90%) and only limited renal excretion (ca. 10% of the dose). In female rats almost equal parts of the test substance were excreted with the urine (ca. 40%) and faeces (ca. 60%). See Table 4-5 under 'Any other information on results incl. tables'.
At 48 hours after dosage, the major part of the radioactivity had been excreted (88 – 96% of the administered dose in males and females). Only a very minor part of the dose was excreted in the time range between 48 and 168 hours after administration. The expiration of 14C-carbon dioxide and other 14C-labelled volatiles was tested with animals for a test period of 48 hours. Less than 0.01% of the administered dose was expired during this sampling period in males and females. This demonstrates the stability of the phenyl-UL-14C label with regard to possible formation of volatile products.

Metabolites identified:

not measured

Table 2: Distribution of radioactivity in organs and tissues of male rats after a single oral administration of 3 mg/kg bw test substance

	Total radioactive residues (TRR) in organs and tissues of male rats [μg a.s. equiv./g wet]
	Animal No. Time of sacrifice (h after administration)
Organ/tissue	650 1 h	651 4 h	652 8 h	653 24 h	654 48 h	655 72 h	656 120 h	657 168 h
Blood	0.702	0.060	0.139	0.027	-	-	-	-
Liver	4.163	1.511	1.577	1.474	1.299	1.258	1.528	1.451
Renal cortex	1.614	0.898	0.860	0.910	0.679	0.636	0.765	0.555
Renal medulla	1.101	0.458	0.556	0.47	0.336	0.348	0.281	0.384
Brown fat	0.200	0.014	0.038	-	-	-	-	-
Perirenal fat	0.063	-	-	-	-	-	-	-
Skeletal muscle	0.053	<LOQ	0.009	< LOQ	-	-	-	-
Myocardium	0.241	0.016	0.040	0.006	-	-	-	-
Lung	0.302	0.030	0.072	0.006	-	-	-	-
Spleen	0.105	0.014	0.036	-	-	-	-	-
Pancreas	0.082	0.008	-	-	-	-	-	-
Bone marrow	0.139	0.017	0.039	0.012	-	-	-	-
Testes	0.152	0.012	0.019	< LOQ	-	-	-	-
Brain	0.011	< LOD	< LOQ	< LOD	-	-	-	-
Spinal cord	0.017	< LOD	< LOQ	-	-	-	-	-
Pituitary gland	0.123	-	0.024	-	-	-	-	-
Pineal body	0.139	-	0.019	-	-	-	-	-
Adrenal gland	0.296	0.018	0.061	0.010	-	-	-	-
Thymus	0.070	0.008	0.017	0.004	-	-	-	-
Thyroid gland	0.220	0.014	0.034	-	-	-	-	-
Salivary gland	0.151	0.010	0.026	-	-	-	-	-
Nasal mucosa	0.049	0.004	0.012	-	-	-	-	-
Skin	0.243	0.013	0.028	-	-	-	-	-
Vitreal body (eye)	0.091	< LOD	< LOQ	< LOD	-	-	-	-

--- organ or tissue usually visible in the rat sections but not discernible in the radioluminograms

LOD/LOQ: limit of detection/limit of quantification

 Table 3: Distribution of radioactivity in organs and tissues of female rats after a single oral administration of 3 mg/kg bw test substance

	Total radioactive residues (TRR) in organs and tissues of male rats [μg a.s. equiv./g wet]
	Animal No. Time of sacrifice (h after administration)
Organ/tissue	659 1 h	660 4 h	661 8 h	662 24 h	663 48 h	664 72 h	665 120 h	666 168 h
Blood	0.417	0.074	0.017	< LOQ	-	-	-	-
Liver	5.162	2.720	2.451	2.266	2.222	1.902	1.580	1.680
Renal cortex	1.916	1.032	1.007	1.088	1.052	1.055	0.700	0.843
Renal medulla	2.699	1.585	1.420	1.368	1.402	1.364	1.072	1.163
Brown fat	0.178	-	-	-	-	-	-	-
Perirenal fat	0.049	-	-	-	-	-	-	-
Skeletal muscle	0.040	0.004	< LOD	-	-	-	-	-
Myocardium	0.143	0.016	< LOQ	< LOD	-	-	-	-
Lung	0.165	0.047	0.009	< LOD	-	-	-	-
Spleen	0.093	0.018	0.004	< LOD	-	-	-	-
Pancreas	0.068	0.011	0.005	< LOD	-	-	-	-
Bone marrow	0.133	0.025	< LOQ	-	-	-	-	-
Ovary	0.182	0.019	-	-	-	-	-	-
Uterus	0.203	-	-	-	-	-	-	-
Brain	0.010	< LOD	< LOD	< LOD	-	-	-	-
Spinal cord	0.011	< LOD	< LOD	-	-	-	-	-
Pituitary gland	0.104	0.013	-	-	-	-	-	-
Pineal body	-	0.006	-	-	-	-	-	-
Adrenal gland	0.238	0.035	0.010	-	-	-	-	-
Thymus	0.053	0.008	< LOQ	-	-	-	-	-
Thyroid gland	0.151	0.021	-	-	-	-	-	-
Salivary gland	0.117	0.012	< LOQ	-	-	-	-	-
Nasal mucosa	0.034	0.006	< LOQ	-	-	-	-	-
Skin	0.148	0.015	-	-	-	-	-	-
Vitreal body (eye)	0.015	< LOD	< LOD	-	-	-	-	-

 --- organ or tissue usually visible in the rat sections but not discernible in the radioluminograms

LOD/LOQ: limit of detection/limit of quantification

Table 4: Total excretion of radioactivity in urine, faeces and expired air of male rats

	Radioactivity in percent of dose administered
Animal No. Time of sacrifice (h after administration)	659 1 h	660 4 h	661 8 h	662 24 h	663 48 h	664 72 h	665 120 h	666 168 h
Expired air					0.006	0.006	0.005	0.005
Urine	2.53	4.68	9.86	9.01	8.45	10.64	8.16	7.17
Faeces	-	-	-	0.15	86.20	86.32	84.17	88.66
Urine + faeces + expired air	2.53	4.68	9.86	9.15	94.65	96.97	92.33	95.83
Total excreted	2.53	4.68	9.86	9.15	94.65	96.97	92.33	95.83

 Table 5: Total excretion of radioactivity in urine, faeces and expired air of female rats

	Radioactivity in percent of dose administered
Animal No. Time of sacrifice (h after administration)	659 1 h	660 4 h	661 8 h	662 24 h	663 48 h	664 72 h	665 120 h	666 168 h
Expired air					0.005	0.007	0.005	0.005
Urine	19.01	24.14	35.23	31.93	34.84	29.92	31.99	37.30
Faeces	-	-	-	52.33	53.46	63.47	58.64	59.42
Urine + faeces + expired air	19.01	24.14	35.23	84.26	88.30	93.40	90.63	96.73
Total excreted	19.01	24.14	35.23	84.26	88.30	93.40	90.63	96.73

 

Reference 2

Endpoint:

basic toxicokinetics in vivo

Remarks:

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

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 04 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

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

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

Dose / conc.:

2 other: mg/kg bw (nominal)

Remarks:

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

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.

Type:

absorption

Results:

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

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)

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.

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.

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 plasma 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 excretion 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.

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: 1.07

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 1st: 0.30

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 1st: 0.07

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 1st: 0.45

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 2nd: 2.37

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 2nd: 2.95

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 2nd: 1.97

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 2nd: 1.41

Key result

Test no.:

#1

Toxicokinetic parameters:

half-life 3rd: 17.80

Key result

Test no.:

#2

Toxicokinetic parameters:

half-life 3rd: 27.30

Key result

Test no.:

#3

Toxicokinetic parameters:

half-life 3rd: 16.00

Key result

Test no.:

#4

Toxicokinetic parameters:

half-life 3rd: 12.30

Key result

Test no.:

#1

Toxicokinetic parameters:

AUC: 3.99

Key result

Test no.:

#2

Toxicokinetic parameters:

AUC: 2.71

Key result

Test no.:

#3

Toxicokinetic parameters:

AUC: 1250

Key result

Test no.:

#4

Toxicokinetic parameters:

AUC: 933

Key result

Test no.:

#1

Toxicokinetic parameters:

Cmax: 3.43

Key result

Test no.:

#2

Toxicokinetic parameters:

Cmax: 1.91

Key result

Test no.:

#3

Toxicokinetic parameters:

Cmax: 277.0

Key result

Test no.:

#4

Toxicokinetic parameters:

Cmax: 284.0

Key result

Test no.:

#1

Toxicokinetic parameters:

Tmax: 0.31

Key result

Test no.:

#2

Toxicokinetic parameters:

Tmax: 0.13

Key result

Test no.:

#3

Toxicokinetic parameters:

Tmax: 1.0

Key result

Test no.:

#4

Toxicokinetic parameters:

Tmax: 0.8

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.

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

 

Reference 3

Endpoint:

basic toxicokinetics in vivo

Remarks:

absorption, distribution, metabolism and excretion in rats following a single oral administration; including bile excretion

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Dec 2004 - 25 Jan 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 04 Apr 1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

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: 9 weeks (males), 12-13 weeks (females)
- Weight at study initiation: males: 185 - 207 g; females: 188 - 219 g (body weight at sacrifice)
- 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, bile 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
- Water: Before surgery, tap water from the local mains supply was given ad libitum. After surgery and during the test, the drinking water was supplied with 10% glucose, 0.5% NaCl and 0.05% KCl.
- Acclimation period: Approximately 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Approximately 23
- Humidity (%): 42 - 55
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

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 8.5 mL (= 4.8 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 12.0 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 mg/kg bw, in a volume of 2 mL.

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

2 other: mg/kg bw (nominal)

Remarks:

Actual dose: 2.0 mg/kg bw (males); 2.05 mg/kg bw(females)

No. of animals per sex per dose / concentration:

8

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, bile; gastrointestinal tract, skin, residual carcass
- Time and frequency of sampling:
Bile: Collected at 0 – 4 h, 4 – 8 h, 8 – 24 h, 24 – 32 h and 32 – 48 h separately for each animal, in test tubes cooled by crushed ice.
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 gastrointestinal tract, skin and residual carcass were retained.
- From how many animals: samples pooled per sex and time period
- Method type(s) for identification: Liquid scintillation counting (blood, bile, urine); combustion/LSC (radioactivity in faeces, gastrointestinal tract, skin, residual carcass).

- Other: The total radioactivity in the test substance and metabolites was determined in bile samples, the excreta (urine and faeces) as well as in blood, gastrointestinal tract, skin and carcass. The metabolism was investigated by radio-HPLC and spectroscopic methods in selected bile and urine samples as well as in faeces extracts.

Cholangiostomy:
The animals were anaesthetised for the cholangiostomy surgery with Narcoren (Merial GmbH, Hallbergmoos, Germany) mixed with Atropine (WDT, Garbsen, Germany) in physiological NaCl solution. An indwelling biliary duodenal catheter was implanted in the bile duct for collection of the bile. The operated zone was relocated into the body by lifting the skin. A small incision was made on the back at the basis of the tail of the animal to pull the bile catheter through. Animals were kept individually in the metabolism cages and the cannula of each animal led through the roof of the cage to permit bile collection into an appropriate weighed container which could be removed and replaced without disturbing the animal. The animals received a diluted analgesic (Metamizol®) during and after surgery by intramuscular injection.

Sacrifice: 48 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.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine, faeces, blood, bile; gastrointestinal tract, skin, residual carcass.
- Time and frequency of sampling:
Bile: Collected at 0 – 4 h, 4 – 8 h, 8 – 24 h, 24 – 32 h and 32 – 48 h separately for each animal, in test tubes cooled by crushed ice.
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 gastrointestinal tract, skin and residual carcass were retained.
- From how many animals: Samples pooled per sex (test 1 = males, test 2 = females) and time period (urine, bile in males, 0-24 h; urine, bile in females, 0-48 h; faeces in males and females, 0-48 h).
- Method type(s) for identification: Liquid scintillation counting (blood, bile, urine); combustion/LSC (radioactivity in faeces, gastrointestinal tract, skin, residual carcass); 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, bile).

- 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 12 – 31 cpm (approximately equal to 12 – 31 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.

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.

Type:

absorption

Results:

92.08% in male rats and 88.27% in female rats

Type:

distribution

Results:

In males 6.36%, and in females 8.23% of the administered dose, respectively, was recovered from the organs and tissues (blood, skin, gastrointestinal tract and carcass)

Type:

metabolism

Results:

In males > 80% of the parent compound was metabolised, in females > 60% 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.

Type:

excretion

Results:

Total elimination: 91.83% for males and 90.69% for females. Main elimination routes males: 10.93% in the urine, 75.44% in the bile and 5.46% in the faeces. Main elimination routes females: 33.66% in the urine, 47.51% in the bile and 9.52% in the faeces.

Type:

other: bile excretion

Results:

52.5% of the administered dose was detected in the bile of the males and 22.4% of the dose of the administered dose was detected in the bile of the females 4 h after dosing

Details on absorption:

The oral absorption rate of the test substance was calculated from the recoveries in the urine, bile and organs/tissues: 92.08% in male rats and 88.27% in female rats. The gastrointestinal tract and skin were excluded because it was not possible to say whether the radioactivity present was due to passive diffusion across the gut was or whether the test substance had not been absorbed.
The test substance absorption rate was considered to be fast because of the quantitatively high radioactivity values detected in the bile of the males (52.5% of the dose) and females (22.4% of the dose) 4 h after dosing. See Table 1 under 'Any other information on results incl. tables'. In both males and females, only a minor part of the dose was not absorbed, but excreted directly via the faeces.

Details on distribution in tissues:

The recovery of radioactivity in the organs and carcass (= sum organs/tissues minus the gastrointestinal tract) at sacrifice was 5.74% for the males and 7.14% for the females. For the gastrointestinal tract, the recovery was 0.63% for the males and 1.09% for the females. The respective values for the skin were below 0.1% for males and females. In the blood (measured in erythrocytes and plasma), the recovery percentage was < 0.01% in males and < 0.03% in females. (See results as equivalent concentrations (C), dose normalised concentrations (CN) and radioactivity (%) in Table 4 under 'Any other information on results incl. tables').

Details on excretion:

The excretion of radioactivity via bile, urine and faeces in males and females is shown in Table 1-2 under 'Any other information on results incl. tables'. Significant sex differences were observed. The elimination of radioactivity via the bile was about 7-times greater than that via urine for male rats. For female rats, elimination of radioactivity via the bile was slightly greater than that via the urine. In both tests, only a minor part of the dose was not absorbed and excreted directly via the faeces. The mean proportions of the administered dose eliminated by the males were 10.93% in the urine, 75.44% in the bile and 5.46% in the faeces. The mean proportions of the administered dose eliminated by the females were 33.66% in the urine, 47.51% in the bile and 9.52% in the faeces. In terms of total elimination (sum of urine, faeces and bile, see Table 3 under 'Any other information on results incl. tables'). The level of elimination of [phenyl-UL-14C]-test substance was similar in male and female rats (91.83% for males and 90.69% for females) 48 hours after administration.

The bile duct cannulation did not decrease the proportion of the radioactivity found in the urines of both genders. The amounts detected in the respective bile samples indicated that nearly the total faecal radioactivity had most likely been absorbed and excreted via the bile and no reabsorption (entero-hepatic cycle) of radioactivity from the faeces occurred.

The mean recovery (measured as radioactivity) in urine, bile and faeces, and in organs and tissues, was 98.20% in males and 98.92% in females.

Metabolites identified:

yes

Details on metabolites:

The HPLC-profiles of urine, bile and faeces samples were very similar for males and females. No typical conjugates for example with glucuronic acid or sulphate were detected in the bile samples. The recovery of parent compound and metabolites is shown in Table 5 (males) and Table 6 (females) under 'Any other information on results incl. tables'.

The identification rate was high and parent compound and metabolites constituted in total 85.35% of the administered dose in males (test 1) and 86.71% in females (test 2). In males, unknown metabolites accounted in total for 0.57% of the dose in the urine, 3.94% in the bile and 0.39% in the faeces extract, respectively. In females, unknown metabolites accounted in total for 1.04% of the dose in the urine, 2.10% in the bile and 0.57% in the faeces extract. No individual unknown metabolite had a higher concentration than 0.7% of the dose in the urine, 0.8% in the bile and 0.3%% in the faeces.

The parent compound was present only at low levels in male rats, with 6.30% detected in bile and excreta. The opposite was the case for female rats, where the results showed significantly higher values (14.08% in the urine and 24.82% in total). Significant sex differences were also seen for the quantity of metabolites detected in the respective samples. The extent of metabolism was greater in males than in females, while higher values were generally found in the bile samples for both males and females. The tetrahydrofurane ring of the molecule was the preferred site for metabolism. No metabolic changes were detected at the phenyl ring and cyclohexyl ring. Parent compound-hydroxy-pentanoic acid (M 4) was the dominant metabolite. The values of all other metabolites were significantly lower. From this group, 5-oxo-THF (M 7), oxo-pentanoic acid (M 5A) and dihydroxy-pentyl (M 3) were identified. Cleavage of the tetrahydrofurane ring leading to the urinary and biliary metabolites benzylic alcohol (M 2) and hydroxy-benzylic alcohol (M 1) was a relatively insignificant reaction, since the values for these metabolites did not exceed 0.84% of the administered dose.
The proposed metabolic pathway is attached under "Attached background material".

Mortality:

Due to poor health conditions after surgery, three animals from each experiment (3 males and 3 females) were sacrificed within the sampling period.

ADME results:

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

Sex (test)	Male (1)	Female (2)
Radioactivity (%) of administered dose (mean values of 5 animals/sex)
Urine	10.93	33.66
Bile	75.44	47.51
Faeces	5.46	9.52
Total excreted	91.83	90.69
Skin	0.030	0.040
Sum of organs/tissues	5.705	7.098
Body without gastrointestinal tract	5.735	7.138
Gastrointestinal tract	0.628	1.092
Total body	6.363	8.230
Balance	98.90	98.92
Radioactivity (%) of dose recovered (mean values of 5 animals/sex)
Urine	11.12	33.93
Bile	76.81	48.55
Faeces	5.56	9.11
Total excreted	93.49	91.59
Skin	0.032	0.040
Sum of organs/tissues	5.845	7.287
Body without gastrointestinal tract	5.877	7.327
Gastrointestinal tract	0.640	1.076
Total body	6.517	8.403
Norm.-factor	1.02	1.02

 Table 2: Time course of radioactivity in excreta of male and female rats following a

single oral administration of 2 mg/kg bw

Sex (test)	Male (1)	Female (2)
Cumulative excretion of radioactivity (%) of administered dose (mean values of 5 animals/sex)
Time (h after administration)
Urine
4	1.86	8.38
8	6.91	12.03
24	10.30	23.52
32	10.52	26.93
48	10.93	33.66
Bile
4	52.50	22.40
8	68.58	35.15
24	74.68	43.13
32	75.15	44.59
48	75.44	47.51
Faeces
24	4.72	6.94
48	5.46	9.52
Sum	91.83	90.69
Cumulative excretion of radioactivity (%) of recovered dose (mean values of 5 animals/sex)
Time (h after administration)
Urine
4	1.94	8.66
8	7.01	12.30
24	10.45	23.47
32	10.68	26.93
48	11.12	33.93
Bile
4	52.89	22.43
8	69.54	35.80
24	76.01	43.90
32	76.51	45.44
48	76.81	48.55
Faeces
24	4.79	6.63
48	5.56	9.11
Norm.-factor	1.02	1.02

 Table 3: Urine, bile and faeces pool samples used for analytics

Sex (test)	Male (1)	Female (2)
Radioactivity (%) of administered dose (pooled)
Urine pool 0 – 24 h	10.30	-
Urine pool 0 – 48 h	-	33.6
Urine not analysed	0.63	-
Bile pool 0 – 24 h	74.68	-
Bile pool 0 – 48 h	-	47.51
Bile not analysed	0.76	-
Faeces pool 0 – 48 h	5.46	9.52

 Table 4: Equivalent concentrations (C), dose normalised concentrations (CN) and radioactivity (%)

of dose administered in organs and tissues of 5 male and 5 female rats 48 h after administration

Mean equivalent concentration C [µg/g]
Sex (test)	Male (1)	Female (2)
Erythrocytes	0.0058	0.0390
Plasma	0.0056	0.0101
Skin	0.0025	0.0038
Carcass	0.1811	0.2194
Gastrointestinal tract	0.0972	0.3008
Body without gastrointestinal tract	0.1278	0.1575
Total in body	0.1269	0.1683
Mean dose normalised concentration CN
Sex (test)	Male (1)	Female (2)
Erythrocytes	0.0029	0.0187
Plasma	0.0028	0.0049
Skin	0.0013	0.0018
Carcass	0.0907	0.1074
Gastrointestinal tract	0.0494	0.1456
Body without gastrointestinal tract	0.0640	0.0 772
Total in body	0.0636	0.0823
Mean radioactivity (%)
Sex (test)	Male (1)	Female (2)
Erythrocytes	0.0039	0.0244
Plasma	0.0030	0.0068
Skin	0.0301	0.0396
Carcass	5.6980	7.0670
Gastrointestinal tract	0.6280	1.0920
Body without gastrointestinal tract	5.7350	7.1380
Total in body	6.3630	8.2300

 Table 5: Quantitative evaluation of parent compound and metabolites in the urine, bile and

faeces of male rats following a single oral administration of 2 mg/kg bw

Sample and sampling period [h after administration]		Urine 0-24	Bile 0-24	Faeces 0-48	Total
		% of dose administered
Residue in analysed samples		10.30	74.68	5.46	90.44
Peak ID	Report name [parent substance]-
M 1	OH-benzylic alcohol	-	0.84	-	0.84
U 2		-	0.36	-	0.36
U 3		0.20	0.55	-	0.75
U 5		0.12	0.82	0.18	1.12
M 2	Benzylic alcohol	0.08	-	-	0.08
U 6		0.08	0.47	-	0.56
M 3	Di-OH-pentyl	0.58	0.23	0.19	1.01
M 4	OH-pentanoic acid	7.69	61.56	0.50	69.75
U 7		-	0.56	-	0.56
U 8		0.17	0.66	-	0.83
M 5	Oxo-pentanoic acid (M 5A)	0.54	3.93	0.07	4.54
U 10		-	0.51	-	0.51
M 7	5-Oxo-THF	0.65	2.06	0.14	2.84
U 11		-	-	0.21	0.21
M 8	Parent compound	0.18	2.13	3.99	6.30
Sum identified		9.73	70.74	4.89	85.35
Sum unknown*		0.57	3.94	0.39	4.91
Subtotal		10.30	74.68	5.28	90.26
Solids		-	-	0.17	0.17
Not analysed faeces sample		-	-	0.01	0.01
Total		10.30	74.68	5.46	90.44
Not analysed urine, bile (24-48 h)		0.63	0.76	-	1.39
Sum total					91.83

*Sum unknown urine: 4, each of them < 0.2% of dose; Sum unknown bile: 7, each of them < 0.82% of dose;

Sum unknown faeces: 2, each of them < 0.21% of dose

Table 6: Quantitative evaluation of parent compound and metabolites in the urine, bile and faeces

of female rats following a single oral administration of 2 mg/kg bw

Sample and sampling period [h after administration]		Urine 0-24	Bile 0-24	Faeces 0-48	Total
		% of dose administered
Residue in analysed samples		33.66	27.51	9.52	90.69
Peak ID	Report name [parent substance]-
M 1	OH-benzylic alcohol	-	0.48	-	0.48
U 2		-	-	-	-
U 3		0.24	0.51	-	0.75
U 4		-	-	-	-
U 5		-	0.49	0.30	0.79
M 2	Benzylic alcohol	0.12	-	-	0.12
U 6		0.08	-	-	0.08
M 3	Di-OH-pentyl	1.73	1.02	0.28	3.03
M 4	OH-pentanoic acid	9.04	36.67	0.21	45.92
U 7		-	0.38	-	0.38
U 8		0.72	0.72	-	1.44
M 5	Oxo-pentanoic acid (M 5A)	1.08	1.88	-	2.96
U 10		-	-	-	-
M 7	5-Oxo-THF	6.57	2.68	0.12	9.37
U 11		-	-	0.27	0.27
M 8	Parent compound	14.08	2.67	8.07	24.82
Sum identified		32.62	45.41	8.68	86.71
Sum unknown*		1.04	2.10	0.57	3.71
Subtotal		33.66	47.51	8.68	86.71
Solids		-	-	0.26	0.26
Not analysed faeces sample		-	-	0.02	0.02
Sum total		33.66	47.51	9.52	90.69

*Sum unknown urine: 3, each of them < 0.2% of dose; Sum unknown bile: 4, each of them < 0.72% of dose;

Sum unknown faeces: 2, each of them < 0.30% of dose

Reference 4

Endpoint:

basic toxicokinetics in vivo

Remarks:

depletion of residues and metabolites in excreta and organs/tissues in the rat following single oral administration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Dec 2005 - 11 Oct 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

other: depletion of residues from plasma, liver and kidney, and metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 04 Apr 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

adopted 22 Jul 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen

Radiolabelling:

yes

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), 12 - 13 weeks (females).
- Weight at study initiation: Males: 193 - 210 g; females: 186 - 215 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): 20 - 23
- Humidity (%): 40 - 55
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous Tragacanth®

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The radiolabelled compound was shipped in solid form. The stock solution was prepared by dissolving the respective sample in a total of 25 mL acetonitrile (2.2 mg/mL). It was stored in a freezer (≤ -20°C) until preparation of the administration suspension. The identity and purity of the parent compound in the stock solution was verified by LC-MS/MS and HPLC and analysis. Two administration suspensions were prepared, one for the male treatment groups and the second for the female treatment groups. The radioactive stock solution (8.12 mL, containing nominally 18 mg test item) was pipetted into a glass flask and concentrated under a gentle stream of nitrogen. The nearly dry residue was suspended in 36 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 purity of the test compound was checked by HPLC. The concentration of the active substance was determined by LSC.

Duration and frequency of treatment / exposure:

Single treatment

Dose / conc.:

5 other: mg/kg bw (nominal dose)

Remarks:

Actual dose: 5.11 mg/kg bw (mean, males); 5.08 mg/kg bw (mean, females)

No. of animals per sex per dose / concentration:

4 (per post-administration sample time)

Control animals:

no

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood (plasma and erythrocytes); liver, kidneys, carcass, skin, gastrointestinal tract.
- Time and frequency of sampling: 0.5, 24 and 168 h post-administration the animals (4/sex/time period) were anesthetised using Pentobarbital-Na and sacrificed and exsanguinated by transection of the cervical blood vessels. The sample collection schedule for urine and faeces per animal are given in Table 1 under "Any other information on materials and methods incl. tables".
Urine: Collected 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 separately for each animal in a cryogenic trap before they were lyophilised (freeze-dried), weighed, and homogenised.
Blood: The collected blood was separated into plasma and blood cells by centrifugation.
Tissues/organs: The skins, gastrointestinal tracts and the residual carcasses 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. Liver and kidneys of the individual rats were weighed and pooled for the extraction of radioactive residues.
- Method type(s) for identification: Liquid scintillation counting (blood, urine); combustion/LSC (radioactivity in faeces, tissues and organs); HPLC (analysis of the stock solution and administration suspension of the test item).

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, blood (plasma and erythrocytes); liver, kidneys, carcass, skin, gastrointestinal tract.
- Time and frequency of sampling: 0.5, 24 and 168 h post-administration the animals (4/sex/time period) were anesthetised using Pentobarbital-Na and sacrificed and exsanguinated by transection of the cervical blood vessels. The sample collection schedule for urine and faeces per animal are given in Table 1 under "Any other information on materials and methods incl. tables".
Urine: Collected 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 separately for each animal in a cryogenic trap before they were lyophilised (freeze-dried), weighed, and homogenised.
Blood: The collected blood was separated into plasma and blood cells by centrifugation.
Tissues/organs: The skins, gastrointestinal tracts and the residual carcasses 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. Liver and kidneys of the individual rats were weighed and pooled for the extraction of radioactive residues.
- From how many animals: 4 animals/sex/time period, samples were pooled.
- Method type(s) for identification: radio-HPLC and LC-MS/MS to identifiy and quantify parent compound and metabolites. Rather small peaks or regions additionally detected in the HPLC-chromatograms of the urine and faeces extracts were characterized according to their elution pattern and described as unknown. Identification of these compounds was not deemed necessary, because their amount of radioactivity was too small (< 1% of the administered dose).
- Limits of detection and quantification: For all samples, the limit of detection (LOD) was predefined on the base of the background radioactivity counting rate which was in a range between 12 – 29 cpm (on average 20 cpm), depending on the instrument used. The LOD was established as twice the average background radioactivity. Single background subtraction and a quench and counting efficiency correction for transformation of gross counts (cpm) into net counts (dpm) were automatically performed by the instruments. Therefore, samples with individually measured values below 20 dpm (after correction for the background radioactivity) were not quantified and labelled as < LOD or LOQ in the respective tables. Only in very rare cases were lower values than 20 dpm accepted. The limit of quantification (LOQ) was set as the level of LOD.

Details on absorption:

In males between 94.2% and 99.9% of the administered dose was recovered, and in females between 91.5% and 103.9% of the administered dose was recovered, measured as the total radioactivity in urine, gastrointestinal tract plus faeces and organs and tissues, at sacrifice (see Table 2 under "Any other information on results incl. tables"). This indicates that the absorption was rapid and extensive.

Details on distribution in tissues:

The total radioactive residues (TRR, expressed as percentage of administered dose) in the organs and tissues at sacrifice are given in Table 2-3 under "Any other information on results incl. tables".
In male rats sacrificed 0.5 h post-dosing (Test #1), 24.1% of the dose was detected in the organs and tissues, 65.5% in the gastrointestinal tract plus faeces and 2.8% in urine. During the first 24 h (Test #2), the percentage of the dose in the organs declined to 3.3% and increased to 9.6% in the urine and 87% in the gastrointestinal tract plus faeces fraction. These values did not change significantly within the whole testing period of 7 days (168 h, Test #3) that indicated fast distribution of the absorbed compound related radioactivity within the body. The balance of radioactivity (% recovered) was 94.1 - 99.9%, indicating that the absorption rate is high. The highest TRR-values in the organs of male rats were detected in the liver and kidney, which are the primary sites for metabolism and excretion. From day 1 to day 7, the values in liver and kidney decreased by 13% and 26%, respectively to levels of 2.42% and 0.11% at day 7. Within the same period the values for erythrocytes and other blood corpuscles, plasma, skin and the carcass decreased to ≤ 0.03%.

In female rats sacrificed 0.5 h post-dosing (Test #4), 22.5% of the dose was detected in the organs and tissues, 55.4% in the gastrointestinal tract plus faeces and 11.4% in urine. During the first day (24 h, Test #5), the percentage of the dose in the organs declined to 3.7% and increased to 38.7% in the urine. No significant change occurred for the gastrointestinal tract plus faeces (56.7%). At day 7 (168 h, Test #6), the values for the urine and organs remained relatively constant whereas a slight increase to 63.7% was observed for the gastrointestinal tract plus faeces fraction. This indicated fast distribution of the absorbed compound related radioactivity within the body. The balance of radioactivity (% recovered) was 91.5 - 103.9%, indicating that the absorption rate is high. The highest TRR-values in the organs of female rats were again detected in liver and kidney, which are the primary sites for metabolism and excretion. From day 1 to day 7, the values in liver and kidney decreased by 3% and 29%, respectively to levels of 2.83% and 0.16% on day 7. Within the same period the values for erythrocytes and other blood corpuscles, plasma, skin and the carcass decreased to ≤ 0.05%.

Details on excretion:

The urinary excretion of the test substance was rapid and more than 94% in males and 99% in females, respectively, of the total urinary radioactivity was detected in the first 24 hours after dosing. Significant differences in the excretion rate between sexes were observed. The amount in female rats (11.38% at 0.5 h, 38.7% at 24 h and 36.78% at 24 h in treatment group 4, 5 and 6, respectively) was about 3 times higher than in males (2.82% at 0.5 h, 9.56% at 24 h and 9.53% at 24 h in treatment group 1, 2 and 3, respectively).

Metabolites identified:

yes

Details on metabolites:

Metabolite identification and quantification (see Table 3-4 under "Any other information on results incl. tables".):
Urine:
- Male rats: The major metabolite was AE 0173473-hydroxy-pentanoic acid, followed by AE 0173473-5-oxo-THF with maximum values of 6.2% and 1.1% of the dose, respectively. The amount of the parent compound did not exceed 1.1% of the dose. The values for all the other identified metabolites (AE 0173473-hydroxy-benzylic alcohol, -benzylic alcohol, -dihydroxypentyl and -oxo-pentanoic acid) were below 1% of the dose. The identification rate was ≥ 94%.
- Female rats: The major metabolites were AE 0173473-hydroxy-pentanoic acid and AE 0173473-5- oxo-THF with maximum values of 3.7% and 3.9% of the dose, respectively followed by AE 0173473-dihydroxy-pentyl (1.2%). The parent compound was by far the most significant component, comprising 27.8% of the dose at day one after the study start. The values for all the other metabolites (AE 0173473-hydroxy-benzylic alcohol, - benzylic alcohol and -oxo-pentanoic acid) were below 1% of the dose and therefore of lesser importance. The identification rate was ≥ 97%.

Plasma:
- Male rats: The major component in the 0.5-h sample was the parent compound, with 1.2% of the total, followed by the metabolites AE 0173473-hydroxy-pentanoic acid (0.09%) and AE 0173473-5-oxo-THF (0.07%). Two other metabolites (AE 0173473-oxo-pentanoic acid and one unknown unpolar compound) were detected, although the amount was very low (< 0.02% of the dose). The identification rate was 99%.
- Female rats: The major component in the 0.5-h sample was the parent compound, with 0.6% of the total dose, followed by the metabolites AE 0173473-5-oxo-THF (0.02%) and AE 0173473-hydroxy-pentanoic acid (0.02%). Only one other unknown unpolar compound was detected at a very low level (< 0.01% of the dose). The identification rate was 99%.

Liver:
- Male rats: The major component was the parent compound, accounting for 8.9% of the dose in the 0.5-h sample. This amount decreased to 1.4% of the dose at the test end (168 h). The major metabolites were AE 0173473-hydroxy-pentanoic acid and AE 0173473-5-oxo-THF, which comprised 2.3% and 1.0% of the dose at 0.5 h after administration, respectively. The concentration of both above-mentioned metabolites declined to 0.3% at 168 hours. The values for all other metabolites (AE 0173473-hydroxy-benzylic alcohol, -benzylic alcohol, -dihydroxypentyl, -oxo-pentanoic acid) were ≤ 0.3% of the dose for all sample times. The identification rate was high for all time points (92% - 95%). The TRR-value was high for the first sampling period (15.4 mg/kg) declining to 2.8 mg/kg after 24 h and 2.7 mg/kg after 168 h. The proportion of the parent compound was 3.8- and 9-times higher compared with the metabolites AE 0173473-hydroxy-pentanoic acid- and -5-oxo-THF, respectively, after 0.5 h. On day 7 (168 h sample time), the parent compound was still the major component and was present at levels greater than both the above-mentioned metabolites by a factor of 4.6 and 4.8, respectively.
- Female rats: The major component was the parent compound, accounting for 11% of the dose in the 0.5-h sample. This amount decreased to 1.9% of the dose at the test end (168 h). The major metabolites were AE 0173473-hydroxy-pentanoic acid and AE 0173473-5-oxo-THF, which comprised 1.4% and 1.2% of the dose at 0.5 h after administration, respectively. The concentration of both above-mentioned metabolites declined to 0.2% and 0.3% at 168 hours, respectively. The values for all the other metabolites (AE 0173473-benzylic alcohol, - dihydroxy-pentyl, -oxo-pentanoic acid) were ≤ 0.3% of the dose for all times. The identification rate was high for all time points in time (93% - 98%). The TRR-value was high for the first sampling period (17.9 mg/kg) and remained on a stable level of about 3 mg/kg from day 1 after administration until test end on day 7. The proportion of the parent compound was 7.6- and 8.9-times higher compared with the metabolites AE 0173473- hydroxy-pentanoic acid- and -5-oxo-THF, respectively, after 0.5 h. On day 7 (168 h sample time), the parent compound was still the major component and was present at levels greater than both the above-mentioned metabolites by a factor of 12.1 and 5.8, respectively.

Kidney:
- Male rats: The major component was the parent compound, which accounted for 0.6% of the dose in the 0.5-h sample. This amount decreased to 0.05% of the dose at the test end (168 h). The major metabolites were AE 0173473-hydroxy-pentanoic acid and AE 0173473-5-oxo-THF, which comprised 0.5% and 0.1% of the dose at 0.5 h after administration, respectively. A decline to 0.03% and 0.01%, respectively at 168 hours was observed for both the above-mentioned metabolites. The values for all the other metabolites (AE 0173473-hydroxy-benzylic alcohol, -benzylic alcohol, -dihydroxy-pentyl, -oxo-pentanoic acid) were ≤ 0.05% of the dose for all times. The identification rate was high for all time points (95% - 99%). The TRR-value was comparatively high for the first sampling period (8.5 mg/kg); decreasing by a factor of 10 to 0.9 mg/kg at day 1 after administration. A further decrease was observed for the 168 h sample (0.67 mg/kg). The proportion of the parent compound was 1.1- and 6.4-times higher compared with the metabolites AE 0173473-hydroxy-pentanoic acid- and -5-oxo-THF, respectively, after 0.5 h. On day 7, the parent compound was still the major component and was present at levels greater than both above-mentioned metabolites by a factor of 1.9 and 4.6, respectively.
- Female rats: The major component was the parent compound, accounting for 0.9% of the dose in the 0.5-h sample. This amount decreased to 0.1% of the dose by the test end (168 h). The major metabolites were AE 0173473-hydroxy-pentanoic acid and AE 0173473-5-oxo-THF, which comprised 0.17% and 0.11% of the dose at 0.5 h after administration, respectively. These concentrations declined to 0.01% at 168 hours, respectively. The values for all the other metabolites (AE 0173473-hydroxy-benzylic alcohol, -benzylic alcohol, -dihydroxy-pentyl, -oxo-pentanoic acid) were ≤ 0.05% of the dose for all times. The identification rate was high for all time points (94% - 99%). The TRR-value was comparatively high for the first sampling period (8.2 mg/kg) which decreased by a factor of 5.6 to 1.5 mg/kg at day 1 after administration. A further decrease was observed for day 7 (1.1 mg/kg). The proportion of the parent compound was 5.6- and 8.5-times higher compared with the metabolites AE 0173473-hydroxy-pentanoic acid- and -5-oxo- THF, respectively, after 0.5 h. On day 7, the parent compound remained the major component and was present at levels greater than both the above-mentioned metabolites by a factor of 7.5 and 8.2, respectively.

Summary:
The identification rate of the metabolites was more than 92% of the total radioactive residue (TRR) for the plasma, liver and kidney samples. The amount of parent compound and metabolites in plasma samples of both sexes were on a very low level (< 1.5% of the administered dose) and decreased during the test period to around the limit of detection after 24 h and below after in the 168 h samples. This indicated a relatively rapid depletion of all radioactive components from the plasma. The major component was the parent compound and prominent metabolites were AE 0173473-hydroxy-pentanoic acid and -5-oxo THF. Only traces of AE 0173473-oxo-pentanoic acid were detected in the plasma of male rats. The amount of the total radioactive residues in the extracts from liver and kidney of both sexes was in the same order of magnitude. The parent compound was by far the most significant component in these samples especially in female rats. As for urine and plasma samples, the prominent metabolites were AE 0173473-hydroxypentanoic acid and -5-oxo THF. All other metabolites such as AE 0173473-hydroxybenzylic alcohol, -benzylic alcohol, -dihydroxy-pentyl and -oxo-pentanoic acid were detected in lower amounts. The metabolism of AE 0173473 in rat follows oxidative processes and was more pronounced in male rats. Reactions took place primarily at the tetrahydrofuran ring of the molecule. The first metabolic reaction was an oxidative and obviously important step at the tetrahydrofuran ring of the molecule by which 5-oxo-THF metabolite was formed. Hydrolytic cleavage of the lactone ring led to the major metabolite AE 0173473-hydroxypentanoic acid. Further changes at the ring-opened structure occurred on a quantitatively lower level. All other reactions including cleavage of the tetrahydrofuran ring were of minor importance. The estimated AE 0173473-oxo-pentyl-urea metabolite (M 3a) was a trace metabolite detected in the urine of female rats by the LC-MS analysis. This compound was not seen in the HPLC-chromatograms that were used for quantification due to an obviously lower sensitivity of this method. It was therefore excluded from quantification.
Based on this information, the conclusion is that the metabolism of the parent compound follows oxidative processes and was more pronounced in male rats. Reactions took place at the tetrahydrofuran ring of the molecule by which 5-oxo- THF metabolite was formed. Hydrolytic cleavage of the lactone ring led to the major metabolite AE 0173473-hydroxy-pentanoic acid. Further changes at the ring-opened structure occurred on a quantitatively lower level. All other reactions including cleavage of the tetrahydrofuran ring are of minor importance.

See proposed metabolic pathway attached under 'Attached background material'.

Table 2: Balance of total radioactivity in urine and organs/tissues of male and female rats following a single oral administration of 5 mg/kg bw test substance

Test #	1	2	3	4	5	6
Sex	Male	Male	Male	Female	Female	Female
Duration [h]	0.5	24	168	0.5	24	168
Radioactivity of dose administered (mean values) [%]
Urine	2.82	9.56	10.15	11.38	38.70	37.16
Skin Sum organs	3.98 24.09	0.01 3.33	0.02 2.56	2.20 22.53	0.01 3.66	0.00 3.04
Body without gastrointestinal tract	28.07	3.34	2.58	24.73	3.68	3.05
Gastrointestinal tract + faeces	65.51	86.98	81.43	55.38	56.73	63.67
Balance	96.39	99.88	94.17	91.49	99.10	103.90

 Table 3: Parent compound and metabolites in urine, plasma, liver and kidney of male rats following a single oral administration of 5 mg/kg bw

	Urine			Plasma			Liver			Kidney
Test #	1	2	3	1	2	3	1	2	3	1	2	3
Duration [h]	0.5	24	168	0.5	24	168	0.5	24	168	0.5	24	168
Sampling period/time [h]	0-0.5	0-24	0-168	0.5	24	168	0.5	24	168	0.5	24	168
% Dose administered	2.82	9.56	10.15	1.398	<0.01	<0.01	13.93	2.90	2.42	1.31	0.13	0.11
Compound/metabolite
U 1	0.02	0.04	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
M 1: OH-benzylic alcohol	0.00	0.04	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.01	0.01	0.00	0.00
U 3	0.05	0.17	0.19	n.d.	n.d.	n.d.	1.11	0.03	0.05	0.02	n.d.	0.00
U 5	0.02	0.06	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.01	n.d.	n.d.	n.d.
M 2: Benzylic alcohol	0.04	0.24	0.28	n.d.	n.d.	n.d.	0.18	0.05	0.06	0.01	n.d.	n.d.
M 3: Di-OH-pentyl	0.86	0.77	0.70	n.d.	n.d.	n.d.	0.16	0.02	0.03	0.05	0.00	0.00
M 4: OH-pentanoic acid	1.84	6.10	6.19	0.09	n.d.	n.d.	2.34	0.16	0.30	0.51	0.02	0.03
U 7/U 8	0.05	0.16	0.15	n.d.	n.d.	n.d.	0.11	0.02	0.03	n.d.	n.d.	n.d.
M 5: Oxo-pentanoic acid	0.11	0.52	0.50	0.01	n.d.	n.d.	0.30	0.05	0.07	0.04	0.00	0.00
U 9	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.11	n.d.	n.d.	n.d.	0.00	n.d.
U 10	0.04	0.10	0.09	n.d.	n.d.	n.d.	0.26	0.07	0.08	n.d.	n.d.	0.00
M 7: 5-Oxo-THF	0.32	1.10	0.95	0.07	n.d.	n.d.	0.99	0.18	0.30	0.09	0.01	0.01
U 13	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
U 11	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.09	0.03	0.03	n.d.	n.d.	n.d.
M 8: parent compound	0.06	0.25	1.11	1.21	n.d.	n.d.	8.86	1.94	1.94	0.57	0.09	0.05
U 12	n.d.	n.d.	n.d.	0.02	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
Identified	2.64	9.03	9.73	1.38	-	-	12.84	2.40	2.19	1.28	0.12	0.10
Unknown	0.18	0.53	0.42	0.02	-	-	0.69	0.15	0.20	0.02	0.00	0.01
Subtotal	2.82	9.56	10.15	1.40	-	-	13.52	2.55	2.38	1.29	0.12	0.10
Solids (post-extraction)	-	-	-	-	-	-	0.41	0.35	0.04	0.02	0.01	0.00
Total	2.82	9.56	10.15	1.40	-	-	13.93	2.90	2.42	1.31	0.13	0.11

* metabolite of parent compound, e.g. parent compound-OH-benzylic acid

n.d. = Not detected

Table 4: Parent compound and metabolites in urine, plasma, liver and kidney of female rats following a single oral administration of 5 mg/kg bw

	Urine			Plasma			Liver			Kidney
Test #	1	2	3	1	2	3	1	2	3	1	2	3
Duration [h]	0.5	24	168	0.5	24	168	0.5	24	168	0.5	24	168
Sampling period/time [h]	0-0.5	0-24	0-168	0.5	24	168	0.5	24	168	0.5	24	168
% Dose administered
Compound/metabolite*
U 1	n.d.	0.04	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
M 1: OH-benzylic alcohol	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.01	0.00	0.00
U 3	0.04	0.13	0.14	n.d.	n.d.	n.d.	0.08	0.04	0.04	0.01	n.d.	0.00
U 5	n.d.	0.04	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
M 2: Benzylic alcohol	0.04	0.22	0.21	n.d.	n.d.	n.d.	n.d.	0.02	0.03	0.01	0.00	0.00
M 3: Di-OH-pentyl	0.39	1.23	1.13	n.d.	n.d.	n.d.	0.26	0.07	0.08	0.04	0.01	0.01
M 4: OH-pentanoic acid	1.03	3.70	3.37	0.02	n.d.	n.d.	1.44	0.13	0.16	0.17	0.01	0.01
U 7/U 8	n.d.	0.26	0.33	n.d.	n.d.	n.d.	n.d.	0.01	0.02	n.d.	n.d.	n.d.
M 5: Oxo-pentanoic acid	0.14	0.62	0.58	0.01	n.d.	n.d.	0.20	0.04	0.04	0.02	0.00	0.00
U 9	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.00	n.d.
U 10	0.17	0.43	0.41	n.d.	n.d.	n.d.	0.12	0.05	0.07	0.01	0.01	0.00
M 7: 5-Oxo-THF	1.27	3.93	3.82	0.02	n.d.	n.d.	1.24	0.27	0.33	0.11	0.01	0.01
U 13	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	0.00	n.d.
U 11	n.d.	0.08	0.10	n.d.	n.d.	n.d.	0.08	0.06	0.04	n.d.	n.d.	n.d.
M 8: parent compound	8.28	27.82	26.97	0.59	<0.01	n.d.	10.98	2.40	1.93	0.93	0.16	0.11
U 12	n.d.	0.20	0.11	0.01	n.d.	n.d.	n.d.	0.01	0.02	n.d.	n.d.	n.d.
Identified	11.17	37.52	36.08	0.63	<0.01	-	14.12	2.93	2.58	1.28	0.20	0.15
Unknown	0.21	1.18	1.08	0.01	-	-	0.29	0.17	0.19	0.02	0.01	0.01
Subtotal	11.38	38.70	37.16	0.64	<0.01	-	14.40	3.10	2.76	1.30	0.21	0.16
Solids (post-extraction)	-	-	-	-	-	-	0.24	0.05	0.07	0.02	0.01	0.00
Total	11.38	38.70	36.16	0.64	<0.01	-	14.64	3.16	2.83	1.32	0.22	0.16

* metabolite of parent compound, e.g. parent compound-OH-benzylic acid

n.d. = Not detected

 

Description of key information

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) 1907/2006 and ‘Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance’ (ECHA, 2017), an assessment of the toxicokinetic behaviour of the target substance is 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 and studies in which the toxicokinetic behaviour of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione was investigated.

Based on ADME studies it was observed that 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione is nearly completely absorbed after oral exposure, therefore, a value of 100% oral absorption is used for the chemical safety assessment. A worst case value of 100% absorption is also used for the dermal and inhalation routes. The test substance is characterized by fast distribution in the whole organism and is then rapidly eliminated via renal and faecal excretion. Based on TK and elimination characteristics no signs of bioaccumulation could be observed. The metabolic pathway of the test substance is shown in the attachment. The test substance was shown to be excreted rapidly; within approximately 48 hours, measured as the total cumulative excretion of urine, faeces and expired air. Male rats excreted the test substance mainly via faeces whereas female rats showed excretion to almost equal parts in urine and faeces.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Additional information

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) 1907/2006 and ‘Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance’ (ECHA, 2017), an assessment of the toxicokinetic behaviour of the target substance is 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 and studies in which the toxicokinetic behaviour of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione was investigated.

2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione is a mono-constituent substance with a molecular weight of 442.91 g/mol. The substance is a solid at 20 °C with a melting point of app. 115 °C at 1013 hPa, water solubility of 64.2 g/L at 20 °C and vapour pressure of 1.8E-10 Pa at 20 °C. The log Pow was estimated to be -1.9 at pH 7 and 1 at pH 4.0 (measured as 1.9 at pH 2) and the pH was 3.37 at 20.9°C (1% pure substance in distilled water).

Absorption

Oral

In general, molecular weights below 500 and log Pow values between -1 and 4 are favourable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (> 1 mg/L). Lipophilic compounds may be taken up by micellar solubilisation by bile salts; this mechanism is important for highly lipophilic compounds (log Pow > 4), in particular for those that are poorly soluble in water (≤ 1 mg/L) as these would otherwise be poorly absorbed (ECHA, 2017).

In an in vivo ADME study performed according to OECD guideline 417, a single dose of 2 and 200 mg/kg bw radiolabelled Phenyl-UL-¹⁴C 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione was administered via gavage to 4 Wistar rats/sex (ADME, 2005). The absorption rate was 94.35% for low-dose males and 94.35% for low-dose females, respectively; while the absorption rate for high-dose males was 97.46% and for high-dose females 87.37%, respectively. The absorption rate was measured as the recovery rate from urine, bile and organs/tissue. The Tmax was 0.13 – 0.31 h in the low-dose animals, and 0.8-1.0 h in the high-dose animals. In other studies performed to assess specific parts of the ADME process, the recovery rate was similarly 91 – 104% of the administered dose; measured as recovery in organs/tissues and excreta, or in excreta alone (residue depletion and metabolism, 2006; ADME bile excretion, 2006; distribution and excretion, 2006). The results of the studies show that the substance is rapidly and almost completely absorbed from the GI-tract.

In several oral repeated dose toxicity studies, treatment-related systemic effects were observed following administration of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione (IUCLID Section 7.5.1; 90-days rat, 2003; 90-days, mouse, 2003; 24-month, rat, 2006). Taking into account the available information, the oral absorption rate is considered to be 100%.

Dermal

The dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Molecular weights below 100 g/mol favour dermal uptake, while for those above 500 g/mol the molecule may be too large. Dermal uptake is anticipated to be low if the water solubility is < 1 mg/L; low to moderate if it is between 1-100 mg/L; and moderate to high if it is between 100-10000 mg/L. Log Pow values in the range of 1 to 4 (values between 2 and 3 are optimal) are favourable for dermal absorption, in particular if water solubility is high. For substances with a log Pow above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Log Pow values above 6 reduce the uptake into the stratum corneum and decrease the rate of transfer from the stratum corneum to the epidermis, thus limiting dermal absorption (ECHA, 2017).

The log Pow is predicted to be > - 1.9 < 1 at the pH of the skin (4.5 - 5.5). The log Pow and molecular weight of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione indicate that the substance is likely to be absorbed at a low to moderate rate via the skin.  QSAR estimation resulted in a dermal absorption of 0.01876 µg/(cm2*h) considering a log Pow of -1.9 and 1.539 µg/(cm²*h) with a log Pow of 1.

In a 28-day repeated dose dermal toxicity study, the NOAEL was considered to be 10 mg/kg bw/day, based on the treatment-related microscopic findings observed in the pancreas, in animals administered 100 and 1000 mg/kg bw/day (key, 2013). The observed adverse effects show that the substance is absorbed through the skin, although it is not possible to quantify the rate of absorption. In conclusion, the dermal absorption rate of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione is considered to be 100%.

Inhalation

2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione is a solid with very low vapour pressure (1.8E-10 Pa at 20 °C), and therefore low volatility. Under normal use and handling conditions, inhalation exposure and availability for respiratory absorption of the substance in the form of vapours, gases, or mists is considered to be negligible (ECHA, 2017). However, the substance may be available for inhalatory absorption after inhalation of aerosols, if the substance is sprayed (e.g. as a formulated product). In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract. Particles deposited in the nasopharyngeal/thoracic region will mainly be cleared from the airways by the mucocilliary mechanism and swallowed. The particle size distribution of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione shows that the average of the median particle size D50 was 26.7 µm while the D10 was 7.7 µm. 90% of the particle volume or particle mass had a lower particle diameter than 100.2 µm (D90). This indicates that a significant fraction of the substance has the potential to be inhaled and will primarily be deposited in the nasopharyngeal/thoracic region. A minor fraction (> 10 < 50%) may penetrate into the alveolar region following exposure via the inhalation route.

No treatment-related systemic effects were observed in the acute inhalation toxicity study (key, 2004). However, as for the oral route treatment-related systemic effects were observed in oral repeated dose toxicity studies, while no treatment-related effects were observed in the acute toxicity study, it is not possible to make a reliable prediction for the inhalation route based on the acute toxicity study. Due to the limited information available a worst-case approach is taken and absorption via inhalation is assumed to be as high as via the oral route.

Distribution and Accumulation

The 4 available studies assessed different aspects of the distribution and potential for accumulation of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione in the rat.

In the ADME study (2005), the distribution of the test substance from the central compartment (plasma) to the organs and tissues was followed by measuring the concentration of the total radioactivity in plasma (0-48 h after administration). 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). 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 plasma 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. 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.

In addition to plasma concentration at different time points, 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).

In the second ADME study (ADME bile excretion, 2006), performed according to OECD guideline 417, a single dose of 2 mg/kg bw radiolabelled Phenyl-UL-¹⁴C 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione was administered via gavage to 8 Wistar rats/sex. The percentage of labelled substance/metabolites was measured 48 h after administration. The recovery of radioactivity in the organs and carcass (= sum organs/tissues minus the gastrointestinal tract) at sacrifice was 5.74% for the males and 7.14% for the females. For the gastrointestinal tract, the recovery was 0.63% for the males and 1.09% for the females. The respective values for the skin were below 0.1% for males and females. In the blood (measured in erythrocytes and plasma), the recovery percentage was < 0.01% in males and < 0.03% in females. These results are similar to those reported in ‘ADME, 2005’. In addition, by cannulating the rats, the recovery in bile could be measured: 52.5% of the administered dose was detected in the bile of the males and 22.4% of the dose of the administered dose was detected in the bile of the females 4 h after dosing.

In the third in vivo study performed according to OECD guideline 417, a single dose of 3 mg/kg bw radiolabelled Phenyl-UL-¹⁴C 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione was administered via gavage to 8 Wistar rats/sex (residue depletion and metabolism, 2006). In a control group 1 rat/sex was administered a single dose of 3 mg/kg bw 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione and treated according to the same protocol. The total radioactive residues (TRR, expressed as percentage of administered dose) was measured in the organs and tissues at sacrifice (0.5, 24 and 168 h). In (male and female) rats sacrificed 0.5 h post-dosing, 22.5 - 24.1% of the dose was detected in the organs and tissues, 55.4 - 65.5% in the gastrointestinal tract plus faeces and 2.8 – 11.4% in urine. During the first 24 h, the percentage of the dose in the organs declined to 3.3 - 3.7% and increased to 9.6 – 38.7% in the urine and 56.7 - 87% in the gastrointestinal tract plus faeces fraction. These values did not change significantly within the whole testing period of 7 days (168 h); indicating fast distribution of the absorbed compound related radioactivity within the body. The highest TRR-values in the organs of the rats were detected in the liver and kidney, which are the primary sites for metabolism and excretion. From day 1 to day 7, the values in liver and kidney decreased by 3 - 13% and 26 - 29%, respectively, to levels of 2.42 – 2.83% and 0.11 – 0.16%. Within the same period the values for erythrocytes and other blood corpuscles, plasma, skin and the carcass decreased to ≤ 0.05%.

In the fourth in vivo study performed according to OECD guideline 417, a single dose of 3 mg/kg bw radiolabelled Phenyl-UL-¹⁴C 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione was administered via gavage to 8 Wistar rats/sex (distribution and excretion, 2006). In a control group 1 rat/sex was administered a single dose of 3 mg/kg bw 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione and treated according to the same protocol. One animal/sex/time point was sacrificed 1, 4, 8, 24, 48, 72, 120 and 168 h after administration. The qualitative distribution of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione in organs and tissues was measured by quantitative whole body autoradiography (QWBA), while the radioluminography (RLG) technique was used to quantify the amount of radiolabelled substance in tissues and organs. This method allows the visualisation of selective enrichments of radioactivity.

In measuring the qualitative distribution, the more intense the blackening of the radioluminograms, the higher is the concentration of the radioactivity in the organs or tissues. One hour after oral administration, the radioactivity was distributed among almost all organs and tissues. The most intense blackening was found in the stomach, the small intestine, the urinary bladder, the liver, and the kidney. Less blackening was found in the blood, heart, and lung (males) and blood, brown (subcutaneous) fat, adrenal gland, uterus, ovary, thyroid, heart, and lung (females), respectively, followed by the other organs and tissues with decreasing intensity of blackening. Little blackening was found in the nasal mucosa, brain, spinal cord, and vitreal body of the eye. These results demonstrate that the test substance was readily absorbed. 4 hours after dosing the radioactivity in all peripheral tissues has decreased considerably with only weak blackening to be seen. Significant blackening could still be detected in the blood, heart, lung, spleen and testes (males), and blood, heart, lung, spleen, adrenal gland, thyroid and ovary (females), respectively. The highest concentrations of radioactivity were present in the excretory organs kidney, liver and in the gastro-intestinal tract for males and females. 8 hours after oral administration, the result was similar to the previous one. The most intense blackening was found in the contents of the large and small intestine as well as in the liver and kidney. 24 hours after administration, the extent and intensity of blackening in most organs and tissues had further decreased in the animals. High blackening was still observed in the contents of the large intestine, the kidney and the liver. Weak blackening could be seen in blood, adrenal gland, heart, and lung of females. In the radioluminograms of the rats sacrificed 48 hours, 72 hours, 120 hours, and 168 hours after oral administration, no blackening above the background could be observed for the organs and tissues, except kidney, liver, and the gastro-intestinal tract. These results demonstrate the fast depletion of test substance-related radioactivity from all peripheral compartments of the rat.

Among the quantitatively analysed organs, tissues, and fluids, the highest equivalent concentrations were observed in the liver, kidney and blood. Moderate peak concentrations were found in the lung and myocardium, brown fat, skin, salivary gland, thyroid, adrenal gland and the reproduction organs. Lower concentrations were reached in all other organs and tissues. The lowest peak concentrations were found in the spinal cord, the brain, and the eye. From peak values, a continuous decrease of radioactivity concentrations by several orders of magnitude, below the limit of quantification was observed for most organs and tissues within 48 hours in male rats and within 24 hours in female rats. High concentrations of radioactivity persisted in kidney and liver of both male and female rats for the whole test period of 7 days indicating significant retention and very slow elimination from these organs.  

The available studies consistently show that the level of parent compound and metabolites is reduced rapidly in most organs (to below the limit of detection within 48 h). In the liver and kidneys the elimination rate is slower, though continuous. 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione and its metabolites will not accumulate in organs or tissues.

Metabolism

In the 2006 ADME study (ADME bile excretion, 2006), the metabolism of 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione was quantified and the major metabolites were identified. In males > 80% of the parent compound was metabolised, in females > 60% was metabolised. The HPLC-profiles of urine, bile and faeces samples were very similar for males and females. No typical conjugates (for example with glucuronic acid or sulphate) were detected in the bile samples. The identification rate was high and parent compound and metabolites constituted in total 85.35% of the administered dose in males (test 1) and 86.71% in females (test 2). In males, unknown metabolites accounted in total for 0.57% of the dose in the urine, 3.94% in the bile and 0.39% in the faeces extract, respectively. In females, unknown metabolites accounted in total for 1.04% of the dose in the urine, 2.10% in the bile and 0.57% in the faeces extract. No individual unknown metabolite had a higher concentration than 0.7% of the dose in the urine, 0.8% in the bile and 0.3%% in the faeces.  

The parent compound was present only at low levels in male rats, with 6.30% detected in bile and excreta. The opposite was the case for female rats, where the results showed significantly higher values (14.08% in the urine and 24.82% in total). Significant sex differences were also seen for the quantity of metabolites detected in the respective samples. The extent of metabolism was greater in males than in females, while higher values were generally found in the bile samples for both males and females. The tetrahydrofurane ring of the molecule was the preferred site for metabolism. No metabolic changes were detected at the phenyl ring and cyclohexyl ring. Parent compound-hydroxy-pentanoic acid was the major metabolite. The values of all other metabolites were significantly lower. From this group, 5-oxo-THF, oxo-pentanoic acid and dihydroxy-pentyl were identified.

The identification of metabolites was elucidated further in a residue depletion and metabolism study performed in rats administered 5 mg/kg bw radioactively labelled 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione via gavage (residue depletion and metabolism, 2006). Plasma samples were drawn 0.5, 24 and 168 h post-administration, along with urine and faeces. The amount of parent compound and major metabolites was quantified in the (plasma, urine and faeces) samples and in liver, kidneys, carcass, skin and GI-tract at the same time intervals. The identification rate of the metabolites was more than 92% of the total radioactive residue (TRR) for the plasma, liver and kidney samples. The amount of parent compound and metabolites in plasma samples of both sexes was very low (< 1.5% of the administered dose) and decreased during the test period to around the limit of detection after 24 h and below after in the 168 h samples. This indicated a relatively rapid depletion of all radioactive components from the plasma. The major component was the parent compound and prominent metabolites were parent compound-hydroxy-pentanoic acid and -5-oxo THF. Only traces of parent compound -oxo-pentanoic acid were detected in the plasma of male rats. The amount of the total radioactive residues in the extracts from liver and kidney of both sexes was in the same order of magnitude. The parent compound was by far the most significant component in these samples especially in female rats. As for urine and plasma samples, the prominent metabolites were parent compound -hydroxypentanoic acid and -5-oxo THF. All other metabolites such as parent compound -hydroxybenzylic alcohol, -benzylic alcohol, -dihydroxy-pentyl and -oxo-pentanoic acid were detected in lower amounts. The metabolism of parent compound in rat follows oxidative processes and was more pronounced in male rats. Reactions took place primarily at the tetrahydrofuran ring of the molecule. The first metabolic reaction was an oxidative and obviously important step at the tetrahydrofuran ring of the molecule by which 5-oxo-THF metabolite was formed. Hydrolytic cleavage of the lactone ring led to the major metabolite parent compound -hydroxypentanoic acid. Further changes at the ring-opened structure occurred on a quantitatively lower level. All other reactions including cleavage of the tetrahydrofuran ring were of minor importance. The estimated parent compound -oxo-pentyl-urea metabolite was a trace metabolite detected in the urine of female rats by the LC-MS analysis. This compound was not seen in the HPLC-chromatograms that were used for quantification due to an obviously lower sensitivity of this method. It was therefore excluded from quantification. Based on this information, the conclusion is that the metabolism of the parent compound follows oxidative processes and is more pronounced in male rats. Reactions took place at the tetrahydrofuran ring of the molecule by which 5-oxo- THF metabolite was formed. Hydrolytic cleavage of the lactone ring led to the major metabolite parent compound -hydroxy-pentanoic acid. Further changes at the ring-opened structure occurred on a quantitatively lower level. All other reactions including cleavage of the tetrahydrofuran ring were shown to be of minor importance. Similar results of the identity and concentration of parent compound and major metabolites isolated from urine and faeces were reported in the 2005 ADME study.

The metabolic pathway proposed for the parent compound (from: residue depletion and metabolism, 2006) is found in the attachments of this section.

Excretion

The test substance was shown to be excreted rapidly; within approximately 48 hours, measured as the total cumulative excretion of urine, faeces and expired air (distribution and excretion, 2006). Significant sex differences were observed. In male rats primarily faecal excretion was observed (ca. 90%) and only limited renal excretion (ca. 10% of the dose). In female rats almost equal parts of the test substance were excreted with the urine (ca. 40%) and faeces (ca. 60%). 48 hours after administration, the major part of the radioactivity had been excreted (88 – 96% of the administered dose in males and females). Only a very minor part of the dose was excreted in the time range between 48 and 168 hours after administration. The expiration of ¹⁴C-carbon dioxide and other ¹⁴C-labelled volatiles was tested with animals for a test period of 48 hours. Less than 0.01% of the administered dose was expired during this sampling period in males and females.

This result is supported by the other available studies (ADME, 2005; ADME bile excretion, 2006; residue depletion and metabolism, 2006). The higher rate of biliary excretion in males was shown in the bile via cannulation (ADME bile excretion, 2006). A difference in excretion between the females administered a low and high dose, but not in the males, was observed in ‘ADME, 2005’. The renal to faecal ratio was 0.11 in low-dose males and 0.56 in low-dose females. 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.

Reference list

ECHA (2017). Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance. Version 3.0, November 2017. European Chemicals Agency, Finland.