N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Jul 2003 to 10 Feb 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Objective of study:

absorption

excretion

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: 94/79/EC

Version / remarks:

1994

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese MAFF

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

EC Number:

410-690-9

EC Name:

N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Cas Number:

103055-07-8

Molecular formula:

C17 H8 Cl2 F8 N2 O3

IUPAC Name:

1-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Alpk:APfSD (Wistar-derived)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 200- 250 g at time of dosing
- Housing: On arrival the animals were housed up to 5 per cage, in multiple rat racks suitable for animals of this strain and the weight range expected during the course of this study.
- Diet: CT1, Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days. Before the start of the study the animals were acclimatised individually to metabolism cages for approximately one day.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 9 Jul 2003 to 10 Feb 2004

Administration / exposure

Route of administration:

other: Oral gavage and intravenous

Vehicle:

polyethylene glycol

Remarks:

PEG 400/ethanol 7/3 (v/v)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Radiolabelled test compound, for the 0.1 mg/kg and 1 mg/kg dose preparations or radiolabelled test compoound, diluted with unlabelled test compound for the 10 mg/kg and 100 mg/kg dose preparations were dissolved in polyethylene glycol 400/ethanol, 7/3, (v/v) at an appropriate concentration to produce the required dose rate. The dose preparations, triplicate aliquots dissolved in ethanol, were analysed for radioactivity content by liquid scintillation counting (LSC).

Duration and frequency of treatment / exposure:

Single

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 mg/kg bw (total dose)

Remarks:

0.2 MBq/kg

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw (total dose)

Remarks:

2 MBq/kg

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

4 MBq/kg

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

4 MBq/kg

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on study design:

- Dose selection rationale: The dose range was selected to be comparable to previous kinetics studies
- The systemic bioavailability of the test substance can be directly determined by the classical method of calculating the ratio of AUC between an oral and intravenous dos

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, whole blood, cage wash and tissues
- Time and frequency of sampling:
Blood: Blood samples were individually collected by tail venepuncture for determination of
radioactivity at 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 288, 384 h after dosing and at
termination. During blood sampling animals were placed over aluminium foil to collect any
excreta produced during blood sampling which was retained and included in the totals
determined for excretion of faeces and urine.
Collection of excreta: Following dosing of the radiolabelled test material, urine and faeces were frozen upon
excretion, by collection over solid carbon dioxide at 24 hourly intervals up to 168 h (0-7 days). The animals were then removed from metabolism cages and housed in stock cages, but were returned to metabolism cages on days 10-11, 14-15, and 19-21 for the collection of excreta as described above. At each collection time, each metabolism cage was rinsed with a suitable volume of water and the washings retained. In addition however, aer collection of the excreta from day 7 and at the end of the study, the metabolism cages were washed with ethanol/water. On days 8-9, 12-13, and 16-18 faeces only were recovered at ambient temperature from the tray papers beneath the stock cages.

- Other:
Preparation method:
Dose preparations: Direct liquid scintillation counting of the diluted solution.
Urine and cage wash: Direct liquid scintillation counting of duplicate weighed samples.
Liver and fat: Homogenisation followed by solubilisation in tissue digestant and liquid scintillation counting of duplicate weighed samples
Brain, testes, kidneys, muscle, lungs and residual carcass: Solubilisation in tissue digestant followed by liquid scintillation counting of duplicate weighed samples.
Faeces: Homogenisation with a small quantity of water before sample oxidation.
Whole blood: Solubilisation in tissue digestant followed by decolorisation with H2O2 and liquid scintillation counting of duplicate weighed samples.
Faecal extracts: Direct liquid scintillation counting of duplicate weighed samples.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling:
Faeces: 0-24 h, 24-48 h, 48-72 h, 72-96 h, 96-120 h, 120-144 h, 144-168 h, 168-192 h, 192-216 h, 216-240 h, 240—264 h, 264-288 h, 288- 312 h, 312-336 h, 336-360 h, 336-384 h, 384- 408 h, 408-432 h, 432-456 h, 456-480 h, 480- 504 h.
Urine: 0-24 h, 24-48 h, 48-72 h, 72-96 h, 96-120 h, 120- 144 h, 144- 168 h, 216-240 h, 240-264 h, 312- 336 h, 336-360 h, 432- 456 h, 456-480 h, 480-504 h urine.
- Method type for identification: LC-MS was carried out using a ion trap mass spectrometer equipped with an electrospray source. The eluent was split between the mass spectrometer and a detector with a ratio of approximately 1:4. The HPLC conditions described in Section 3.4 were used for LC-MS. Negative ion spectra were recorded. LC-MS data were acquired and analysed


ANALYTICAL METHOD
The radiochemical purity of the radiolabelled test substance was determined by HPLC and thin layer chromatography (TLC) prior to dose preparation. The radiochemical purity of the test substance in the dose preparation was also determined by HPLC and TLC after dosing to establish the stability of the test substance in the dose preparation.

- HPLC Method 1, for analysis of the test substance
Column packing: Altima (C18), 5µ
Dimensions: 150 mm x 4.6 mm
Mobile phase A: H2O containing 0.1% formic acid
Mobile phase B: CH3CN containing 0.1% formic acid
Gradient series: Initially 40% A and 60% B, then to 35% A and 65% B over 9 minutes, then to 0% A and 100% B over 2 minutes, held for 2 minutes then back to 40% A and 60% B over 2 minutes
Flow rate: 1 mL/min
Column temperature: Ambient
UV detection: 258 nm
Radiochemical detection: Packard Flo-One with 500 µL liquid cell

- HPLC method 2, for metabolite analysis
Column packing: Altima (C18) 5µ
Dimensions: 150 mm x 4.6 mm
Mobile phase A: H2O containing 0.1% formic acid
Mobile phase B: CH3CN containing 0.1% formic acid
Gradient series: Initially 40% A and 60% B, then to 35% A and 65% B over 9 minutes, then to 0% A and 100% B over 2 minutes, held for 5 minutes then back to 40% A and 60% B over 2 minutes
Flow rate: 1 mL/min
Column temperature: Ambient
UV detection: 254 nm
Radiochemical detection: Packard Flo-One with 500 µL liquid cell

TLC was performed using:
Solvent system 1: dichloromethane - Silica gel plates (60F254)
Solvent system 2: methanol: dichloromethane 2:8 (v/v) - Silica gel plates (60F254)

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The area under the blood concentration time curves (AUC) over 120 hours after dosing, for both dose routes, clearly show that for dose levels between 0.1 and 10mg/kg, systemic exposure is directly proportional to the administered dose. The systemic bioavailability of lufenuron can be directly determined by the classical method of calculating the ratio of AUC between an oral and intravenous dose. This gives a value of approximately 70% for the 0.1 and 10 mg/kg dose levels

Details on distribution in tissues:

At termination of the study the mean total proportion of administered radioactivity present in tissues and carcass following oral dosing at 0.1, 1.0, 10, or 10 mg/kg was 21.0, 23.6, 14.8 and 5.2% respectively. The mean total proportion of dose present in tissues following an intravenous dose of 0.1 or 10 mg/kg was 32.0 and 34.7% respectively.

Details on excretion:

Over the 21 day duration of the study the mean total proportion of administered radioactivity excreted in urine following oral dosing at 0.1, 1.0, 10, or 100 mg/kg was 0.6, 0.5, 0.6 and 0.2% respectively, whilst faecal excretion accounted for 80.6, 72.9, 80.4 and 91.9% respectively. The mean total percentage of dose excreted in urine following an intravenous dose of 0.1 or 10 mg/kg was 0.6 and 0.8% respectively, whilst faecal excretion accounted for 66.5 and 62.2% respectively. A characteristic feature of faecal excretion was the slow, sustained period of elimination. Faecal excretion was still measurable 21 days following administration of a single oral or intravenous dose, irrespective of dose level

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

Table 1. Excretion of test substance by rats over 21 days following a single oral or intravenous dose of [14C]-test substance (Results are expressed as percentages of administered radioactivity)

Hours after dosing	Oral								Intravenous
	0.1 mg/kg		1 mg/kg		10 mg/kg		100 mg/kg		0.1 mg/kg		10 mg/kg
	Urine	Faeces	Urine	Faeces	Urine	Faeces	Urine	Faeces	Urine	Faeces	Urine	Faeces
24	0.13	32.76	0.10	31.05	0.11	40.76	0.04	77.71	0.13	10.72	0.14	6.99
48	0.09	8.54	0.07	6.34	0.09	8.58	0.03	6.91	0.09	6.76	0.13	6.11
72	0.08	5.52	0.08	3.43	0.07	3.15	0.02	0.98	0.06	5.67	0.09	4.57
96	0.06	4.61	0.05	3.97	0.07	2.50	0.01	0.69	0.06	5.10	0.07	4.07
120	0.05	2.91	0.03	2.78	0.05	3.06	0.02	0.52	0.05	4.18	0.05	4.03
144	0.05	3.45	0.04	2.72	0.04	2.61	0.01	0.51	0.05	3.64	0.06	3.59
168	0.04	2.65	0.02	2.54	0.03	2.39	0.01	0.46	0.04	3.43	0.04	3.28
192	n.s.	2.33	n.s.	2.09	n.s.	2.25	n.s.	0.40	n.s.	3.42	n.s.	3.01
216	n.s.	2.00	n.s.	1.78	n.s.	1.76	n.s.	0.38	n.s.	2.51	n.s.	2.81
240	<0.02	1.83	0.02	1.91	0.02	1.54	0.01	0.35	<0.03	2.30	0.04	2.74
264	0.03	1.84	0.02	1.56	0.02	1.62	0.01	0.33	0.03	2.43	0.04	2.64
288	n.s.	1.57	n.s.	1.61	n.s.	1.50	n.s.	0.33	n.s.	2.20	n.s.	2.23
312	n.s.	1.51	n.s.	1.54	n.s.	1.28	n.s.	0.29	n.s.	1.92	n.s.	1.99
336	<0.02	1.38	0.02	1.50	0.02	1.19	<0.01	0.30	<0.03	1.90	0.03	2.23
360	<0.02	1.33	0.02	1.31	0.02	1.12	<0.01	0.28	<0.02	1.71	0.03	1.92
384	n.s.	1.22	n.s.	1.34	n.s.	1.03	n.s.	0.27	n.s.	1.77	n.s.	1.94
408	n.s.	1.12	n.s.	1.14	n.s.	0.90	n.s.	0.24	n.s.	1.43	n.s.	1.72
432	n.s.	1.07	n.s.	1.08	n.s.	0.81	n.s.	0.24	n.s.	1.43	n.s.	1.63
456	<0.02	1.01	0.02	1.08	0.02	0.77	<0.01	0.22	<0.02	1.26	0.03	1.59
480	<0.02	0.95	0.01	1.03	0.01	0.71	<0.01	0.22	<0.03	1.29	0.03	1.46
504	<0.02	0.97	0.01	1.16	0.01	0.84	<0.01	0.25	0.02	1.43	0.03	1.65
0-504	0.64	80.59	0.51	72.93	0.57	80.37	0.17	91.87	0.64	66.50	0.82	62.23

n.s. No sample of urine available during the housing of rats in stock cages.

The highest residues, expressed as μg equivalents test substance/g, were present in fat. Progressively lower residues were found in the residual carcass (in part attributable to fat depots), liver and kidneys.

Table 2. Tissue residues of radioactivity twenty one days after a single oral dose of [14C] - test substance (Results are expressed as μg equivalents test substance/g)

Tissue	Oral				Intravenous
	0.1 mg/kg	1.0 mg/kg	10 mg/kg	100 mg/kg	0.1 mg/kg	10 mg/kg
Brain	<0.001	0.006	0.038	0.112	<0.001	0.079
Fat	0.116	1.407	8.712	26.200	0.162	18.950
Testes	0.001	0.017	0.132	0.483	0.002	0.295
Kidneys	0.006	0.067	0.412	1.336	0.008	0.915
Muscle (skeletal)	0.004	0.040	0.202	0.794	0.004	0.462
Liver	0.007	0.085	0.534	1.745	0.010	1.187
Residual Carcass	0.015	0.172	0.953	3.586	0.023	2.429
Lungs	0.002	0.040	0.352	0.758	0.004	0.614

At termination of the study the mean total proportion of administered radioactivity present in tissues and carcass following oral dosing at 0.1, 1.0, 10, or 10 mg/kg was 21.0, 23.6, 14.8 and 5.2% respectively. The mean total proportion of dose present in tissues following an intravenous dose of 0.1 or 10 mg/kg was 32.0 and 34.7% respectively

Table 3. Tissue residues of radioactivity twenty one days after a single oral dose of [14C] - test substance (Results are expressed as percentages of administered radioactivity

Tissue	Oral				Intravenous
	0.1 mg/kg	1.0 mg/kg	10 mg/kg	100 mg/kg	0.1 mg/kg	10 mg/kg
Brain	<0.01	<0.01	<0.01	<0.01	<0.01	0.01
Fat	1.04	1.20	0.84	0.20	1.47	2.10
Testes	0.02	0.02	0.02	0.01	0.03	0.04
Kidneys	0.07	0.07	0.05	0.01	0.08	0.10
Muscle (skeletal)	0.02	0.02	0.01	<0.01	0.02	0.03
Liver	0.48	0.54	0.41	0.12	0.62	0.78
Residual Carcass	19.34	21.66	13.47	4.82	29.76	31.63
Lungs	0.02	0.03	0.03	0.01	0.03	0.05
Total	21.00	23.55	14.83	5.17	32.02	34.73

The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral dosing at 0.1, 1.0, 10, or 10 mg/kg were 102.9, 97.2, 96.0 and 97.4% respectively. The total mean percentage recoveries of dose following a 0.1 or 10 mg/kg intravenous dose were 99.8 and 98.1% respectively. Hence, all recoveries were considered to be very good, despite the long duration of each experiment.

Table 4. Summary of excretion and tissue distribution of test substance by rats over twenty one following a single oral or intravenous dose of [14C]-test substance (Results are expressed as percentages of administered radioactivity)

	Oral				Intravenous
	0.1 mg/kg	1.0 mg/kg	10 mg/kg	100 mg/kg	0.1 mg/kg	10 mg/kg
Urine	0.64	0.51	0.57	0.17	0.64	0.82
Faeces	80.59	72.93	80.37	91.87	66.50	62.23
Cage Wash	0.69	0.24	0.29	0.22	0.61	0.33
Carcass	21.00	23.55	14.83	5.17	32.03	34.73
Total	102.93	97.23	96.05	97.43	99.77	98.11

Table 5. Blood residues of radioactivity over a time course and terminal blood and plasma residues after a single oral or intravenous dose of [14C]-test substance (Results are expressed as μg equivalents test substance/g).

Hours after dosing	Oral				Intravenous
	0.1 mg/kg	1.0 mg/kg	10 mg/kg	100 mg/kg	0.1 mg/kg	10 mg/kg
1	0.002	0.023	0.116	0.252	N.S.	1.862
2	0.003	0.055	0.199	0.814	0.017	1.907
4	0.007	0.071	0.609	1.026	0.015	1.376
8	0.008	0.097	0.887	1.341	0.010	1.081
12	0.007	0.071	0.527	1.161	0.007	0.774
24	0.005	0.050	0.281	1.185	0.006	0.654
48	0.003	0.030	0.307	0.611	0.004	0.407
72	0.002	0.026	0.292	0.401	0.003	0.382
96	<LOD	0.024	0.429	0.556	0.003	0.327
120	0.002	0.024	0.131	0.487	0.003	0.308
144	<LOD	0.017	0.171	0.307	0.002	0.270
168	<LOD	0.016	0.174	0.257	0.002	0.222
288	<LOD	0.012	0.110	0.240	<LOD	0.174
384	<LOD	0.009	0.052	0.201	<LOD	0.127
504	<LOD	0.006	0.044	0.130	<LOD	0.086
504 (plasma)	<LOD	0.008	0.060	0.167	0.001	0.109

The mean terminal half-life of faecal excretion was 256 hours following oral dosing and 232 hours following intravenous dosing.

Table 6. Terminal half-life for excretion of test substance following a single oral or intravenous dose of [14C]-test substance

Nominal dose, route	Urinary terminal half-life (hours)	Faecal terminal half-life (hours)
0.1 mg/kg, oral gavage	454	255
1.0 mg/kg, oral gavage	452	265
10 mg/kg, oral gavage	348	195
100 mg/kg, oral gavage	238	308
0.1 mg/kg, intravenous.	346	197
10 mg/kg, intravenous	382	267

The blood AUC (0-120 h) increased with dose over the range 0.1-100 mg/kg. At the highest oral dose level, evidence of saturation was observed and the AUC was no longer proportional to the administered dose. Saturation was also evident in the relationship of blood AUC (0-inf.) to dose. Hence, from the AUC values determined for blood, the dose response does not appear to be linear following oral gavage dosing at the highest dose level.

Table 7. Derivation of oral absorption values using comparison of oral & intravenous kinetics

		oral	Intravenous	Oral/intravenous = % absorption
0.1 mg/kg	AUC	0.40	0.56	71
10 mg/kg	AUC	41.25	60.69	68
0.1 mg/kg	% dose in urine	0.64	0.64	100
10 mg/kg	% dose in urine	0.57	0.82	70

Applicant's summary and conclusion

Conclusions:

Following a single oral dose of 0.1, 1.0, 10 or 100 mg [14C]-test substance/kg or a single intravenous dose of 0.1 or 10 mg [14C]-test substance/kg to male rats, the main route of excretion was via faeces, with the test substance being excreted as unchanged parent test substance. The rate of excretion was slow over the 21 day time course of this study. The systemic bioavailability of the test substance can be directly determined by the classical method of calculating the ratio of AUC between an oral and intravenous dose. This gives a value of approximately 70% for the 0.1 and 10mg/kg dose levels

Executive summary:

The excretion and the tissue distribution was investigated in groups of 4 male rats after a single oral dose of [14C]- test substance at 0.1, 1.0, 10 or 100 mg/kg or a single intravenous dose of [14C]-test substance at 0.1 or 10 mg/kg according to OECD TG 417 and GLP principles. Subsequently faeces were collected daily on days 8-21 following dosing and urine was collected on days 10-11, 14-15 and 19-21. Blood samples were taken at time points throughout the 21 day duration of the study. Radioactivity present in excreta and blood was quantified. After 21 days the animals were terminated and selected tissues were removed for determination of radioactivity. The profile of radioactivity present in excreta was also examined.

Results showed that over the duration of the study the mean total proportion of administered radioactivity excreted in urine following oral gavage dosing at 0.1, 1.0, 10, or 100 mg/kg was 0.6, 0.5, 0.6 and 0.2% respectively while faecal excretion accounted for 80.6, 72.9, 80.4 and 91.9% respectively. The mean total proportion of administered radioactivity excreted in urine following i.v. dosing at 0.1, or 10 mg/kg was 0.6 and 0.8% respectively, faecal excretion accounted for 66.5 and 62.2% respectively. A characteristic of the faecal excretion was the sustained period of excretion. Faecal excretion was still measurable 21 days after the administration of a single oral gavage dose of the test substance. At termination of the study the mean total proportion of administered radioactivity present in tissues and carcass following oral gavage dosing at 0.1, 1.0, 10, or 10 mg/kg was 21.0, 23.6, 14.8 and 5.2% respectively. The mean total proportion of administered radioactivity present in tissues following i.v. dosing at 0.1, or 10 mg/kg was 32.0 and 34.7% respectively. Radioactivity was mainly located in the residual carcass, and to a much lower extent, in fat. The total mean percentage recoveries of administered radioactivity including excreta tissues and residual carcasses following oral gavage dosing at 0.1, 1.0, 10, or 10 mg/kg were 102.9, 97.2, 96.0 and 97.4% respectively. The total mean percentage recoveries of administered radioactivity including excreta tissues and residual carcasses following i.v. dosing were 99.8 and 98.1% respectively. One major component was identified in the faecal extracts and this was identified as parent test substance. The levels of administered radioactivity excreted in urine were very low (<1% of administered radioactivity) and metabolite identification was not performed on these samples. From the AUC values determined for blood, the dose response does not appear to be linear following oral gavage dosing at the highest dose level. The area under the blood concentration time curves (AUC) over 120 hours after dosing, for both dose routes, clearly show that for dose levels between 0.1 and 10mg/kg, systemic exposure is directly proportional to the administered dose. The systemic bioavailability of the test substance can be directly determined by the classical method of calculating the ratio of AUC between an oral and intravenous dose. This gives a value of approximately 70% for the 0.1 and 10mg/kg dose levels

In conclusion, following a single oral dose of 0.1, 1.0, 10 or 100 mg [14C]-test substance/kg or a single intravenous dose of 0.1 or 10 mg [14C]-test substance/kg to male rats, the main route of excretion was via faeces, with the test substance being excreted as unchanged parent test substance. The rate of excretion was slow over the 21 day time course of this study. The systemic bioavailability of the test substance can be directly determined by the classical method of calculating the ratio of AUC between an oral and intravenous dose. This gives a value of approximately 70% for the 0.1 and 10 mg/kg dose levels.