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

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

Objective of study:

absorption

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Qualifier:

equivalent or similar to guideline

Guideline:

other: 94/79/EC

Version / remarks:

1994

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

1998

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese MAFF

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

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

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

Dose / conc.:

0.1 mg/kg bw (total dose)

Remarks:

0.2 MBq/kg

Dose / conc.:

1 mg/kg bw (total dose)

Remarks:

2 MBq/kg

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

4 MBq/kg

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)

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

Metabolites identified:

no

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

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.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Dec 2002 to 16 Apr 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

other: Rat and human skin membrane

Sex:

not specified

Type of coverage:

open

Vehicle:

water

Duration of exposure:

The skin was exposed to the test preparations for 24 hours during which time samples of receptor fluid were taken at 1 hour intervals for the initial 6 hours and at 2 hourly intervals thereafter to allow adequate characterisation of the absorption profile.

Doses:

- 0.9 µg/cm2 (Low dose)
- 455 µg/cm2 (High dose)

Details on study design:

Skin preparations: Epidermal membranes were prepared from human and rat whole skin. Eight healthy male HanBrl:WIST (SPF) rats, approximately 9 weeks old, were sacrificed and the fur on the dorsal site clipped and the full thickness skin excised. These skin samples were wrapped in aluminium foil and stored at -18ºC. Abdominal cadaver skin from a Caucasian donor was used and the subcutaneous fat was removed and the skin stored at approximately -18ºC. The skin samples were allowed to thaw at room temperature and subcutaneous fat carefully removed from the full thickness skin and pieces of about 4 x 5 cm2 were stretched evenly over a cork block, with stratum corneum uppermost. Skin sections of about 200 μm thickness were cut off the top using a dermatone. The skin sections were cut in pieces (approx. 1.8 x 1.8 cm2) and mounted in diffusion cells.

Details on in vitro test system (if applicable):

Diffusion cell: Diffusion of the test substance into and across the skin to a receptor fluid was measured using glass diffusion cells in which the epidermis formed a horizontal membrane and provided an application area of 0.64 cm2.

Receptor fluid: The receptor fluid (50% ethanol in water) was chosen to ensure that the test substance would freely partition into this from the skin membrane and never reach a concentration that would limit its diffusion. The receptor fluid was delivered at a flow rate of approximately 3mL/hour.

Skin preparation integrity: The integrity of the membranes was checked by measurement of the electrical resistance across the skin. Only those membranes with an acceptable resistance, thereby showing that they were intact, were used on the study.

Test substance: Two dose levels were used: the low dose level Al reflects a typical concentration recommended for use, e.g. pome and stone fruits (5 -10 g a.i./100 1), while the high dose level A2 represents the concentration of active ingredient in the undiluted formulation (50 g a.i./l). The doses were prepared as close to the time of application as was practicable and were analysed to confirm their suitability for use in the study. Application to the skin: A 6μL aliquot of the application solution/emulsion was applied manually to each skin membrane preparation which was mounted in the diffusion cells
between the donor and receptor chamber, 6 or 7 cells per species and dose level. The donor chamber was left open (non-occluded conditions).

Temperature: Throughout the experiment the receptor fluid was stirred and the membranes were maintained at a temperature of 31 ± 1°C in a water bath.
Duration of exposure and sampling: The skin was exposed to the test preparations for 24 hours during which time samples of receptor fluid were taken at 1 hour intervals for the initial 6 hours and at 2 hourly intervals thereafter to allow adequate characterisation of the absorption profile.

Terminal procedures: Twenty four hours after application the skin membrane surface was rinsed with ethanol (10 mL) and the radioactivity in the skin rinse was determined by liquid scintillation counting (LSC). The skin membrane was removed from the in-line cells and dissolved in tissue solubilizer prior to LSC. The cells were finally washed with ethanol (250 mL) and the radioactivity in the cell wash determined by LSC.

Analysis: Radioactivity was measured by Liquid Scintillation Counting (LSC) equipped for computing quench-corrected disintegrations per minute (dpm). Liquid specimens, i.e. perfusate, skin rinse, cell wash, were added directly to scintillation mixture Irga-Safe plus for the measurement of radioactivity. The radioactivity in the skin membranes was determined after digestion in tissue solubiliser (4 mL). The digested specimens were neutralized with hydrochloric acid (1 mL, 4N) and mixed with Irga-Safe plus (ca. 15 mL) prior to LSC. Background values were measured with each specimen sequence using the respective scintillation mixture without any specimens. The pattern of radioactivity on thin layer plates was detected by using a Imager. The TLC plates were exposed for an appropriate time interval. Further processing of the images including quantification of the radioactive zones was performed on the Imager software.

Definition of absorbed test material: The absorbed (systemically available) dose is considered to be the test material detected in the receptor fluid. Material removed from the surface of the epidermis by the washing procedure is regarded as unabsorbed. The test material recovered from the epidermis at the end of the exposure is also considered to be unabsorbed, although it is recognised that a proportion of this material may be absorbed beyond the duration of the exposure investigated in this study. In vivo, the majority of the dose in the epidermis, especially that recovered from the stratum corneum, would eventually be lost by desquamation.

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Key result

Time point:

24 h

Dose:

Low dose: 0.9 µg/cm2

Parameter:

percentage

Absorption:

12 %

Remarks on result:

other: Human skin membrane

Remarks:

Sum of receptor fluid+ skin sample +(SD*k)

Key result

Time point:

24 h

Dose:

High dose: 455 µg/cm2

Parameter:

percentage

Absorption:

3.9 %

Remarks on result:

other: Human skin membrane

Remarks:

Sum of receptor fluid+ skin sample +(SD*k)

Time point:

24 h

Dose:

Low dose: 0.9 µg/cm2

Parameter:

percentage

Absorption:

9.2 %

Remarks on result:

other: Rat skin membrane

Remarks:

Sum of receptor fluid+ skin sample +(SD*k)

Time point:

24 h

Dose:

High dose: 455 µg/cm2

Parameter:

percentage

Absorption:

21.7 %

Remarks on result:

other: Rat skin membrane

Remarks:

Sum of receptor fluid+ skin sample +(SD*k)

Results:

Within 24 hours the portion of radiolabelled test substance penetrating through rat skin membranes accounted for 1.2 % and 6.4 % of the low and high dose, respectively. The human skin membranes showed a significantly lower permeability to the test substance than the rat skin membranes. At the low dose level all determined values in the perfusates were below the limit of determination (LQ) and at the high dose level only 0.3 % of the dose penetrated through human skin membranes within 24 hours of exposure. A distinct species difference in respect to the penetration of the test substance is also reflected by the flux. The flux at steady-state conditions was determined to be 0.0009 and 1.707.tg/cm2/h through rat skin membranes at the low and high dose level, respectively. For the human skin membranes the flux could be calculated only for the high dose level, accounting for 0.106 pg/cm2/h. For the low dose level the calculation of the flux was not applicable, since all values were below the limit of determination. Making a conservative assumption of the flux for the low dose level based on the LQ values (0.0004 μg/cm2/h) the human/rat ratio of the flux was at least 1:2 for this low dose level. For the high dose level, for which the flux for both species was able to be more accurately determined, the human/rat ratio was about 1:16

Table 1. Summary of absorption of the test substance through rat skin membrane

Test system	Rat skin membrane
Dose level	A1		A2
Applied dose (µg cm-2)	0.9		455
Applied volume (µL)	6		6
Application area (cm2)	0.64		0.64
Concentration (mg cm-3)	0.09		48.58
Penetration within	% of dose	µg cm-2	% of dose	µg cm-2
6 hours	0.20*	<0.01*	0.98	4.47
12 hours	0.47*	<0.01*	3.13	14.25
24 hours	1.15	0.01*	6.38	29.07
Flux (µg /cm2/hour)	0.009**		1.707

* Most values of the penetration per time interval were below the LQ value

** Flux has been calculated only using the 3 cells with values above the LQ

Rat skin membrane

After application of the test substance at the low dose level Al, which represents the typical concentration recommended for field applications, only 1.2 % of the applied dose penetrated through the rat skin membrane within 24 hours. At the high dose level (undiluted formulation) the portion penetrating through the skin membrane accounted for 6.4 % of the high dose. Due to the very low penetration rate, most of the determined values at the low dose level were below the limit of determination (LQ) for the first time intervals (0-12 hours). For the time intervals between 12 and 24 hours only three cells showed values above the LQ and therefore the flux was calculated only for these three cells. The flux, which reflects the penetration rate under steady-state conditions, was calculated to be 0.0009 tg/cm2/h for the

low dose level Al (time interval for steady-state conditions: 8 to 24 hours). At the high dose level A2, the flux was calculated to be 1.707 ig/cm2/h (steady-state conditions: 3 to 24 hours).

Human Skin Membrane

At the low dose level the penetration through human skin membrane was very low. All determined values were at or below the limit of determination (LQ), therefore the calculation of the flux was not applicable. At the high dose level only 0.3 % of dose penetrated through the human skin membrane within 24 hours of exposure. The calculated flux, under steady state conditions, accounted for 0.106.tg/cm2/h. The time intervals for steady state condition were between 8 and 24 hours after administration

Table 2. Summary of absorption of the test substance through human skin membrane

Test system	Rat skin membrane
Dose level	A1		A2
Applied dose (µg cm-2)	0.9		455
Applied volume (µL)	6		6
Application area (cm2)	0.64		0.64
Concentration (mg cm-3)	0.09		48.58
Penetration within	% of dose	µg cm-2	% of dose	µg cm-2
6 hours	0.21*	<0.01*	0.02*	0.08*
12 hours	0.35*	<0.01*	0.09	0.41
24 hours	0.69*	0.01*	0.34	1.56
Flux (µg /cm2/hour)			0.106

* Most values of the penetration per time interval were below the LQ value

** Flux has been calculated only using the 3 cells with values above the LQ

At the end of the experiments, the total recovery of radioactivity was determined. For the Group Q 1 (rat skin membrane) the mean recovery was 95 % at the low dose level Al and 100 % of the applied radioactivity at the high dose level A2. he mean recovery for the experiments of Group Q2 (human skin membrane) was 96 % and 100 % at the low and high dose level, respectively. However, due to the very low amount of total applied radioactivity (< 100'000 dpm) and the small application volume of the emulsion (6 μL) the total recovery for the low dose level showed a high range of variation. The recovered radioactivity at the end of exposure in the skin rinse, skin membrane, and cell wash is summarized in the table below.

Table 3. Summary of recovered radioactivity at the end of exposure

Percentage of Dose Recovered (%)
Test system	Rat skin membrane Group Q1		Human skin membrane Group Q2
Dose level	A1	A2	A1	A2
Applied dose (µg cm-2)	0.9	455	0.9	455
Perfusates 0-24 hour	1.15	6.38	0.69	0.34
Remaining dose Cellwash Skin rinse Skin membrane Subtotal	2.25 87.76 4.04 94.05	1.94 80.26 11.63 93.84	3.07 87.58 4.40 95.05	0.78 96.38 2.41 99.57
Recovery	95.20	100.22	95.74	99.91

Aliquots of the skin rinse were pooled according to species and dose level. The pools were analysed by TLC. The test substance amounted to more than 97 % of the radioactivity present in the skin rinses for both dose levels and both species. Hence, it is concluded that the test substance remained unchanged during the 24 hours of exposure on the skin membrane.

Table 4. Recovery of radioactivity following application of [14C]-test substance to rat skin membrane (Group Q1, dose level A1)

Dose applied	[% of Dose] 0.9 µg/cm2
Cell No.	Cell 2	Cell 3	Cell 4	Cell 5	Cell 6	Cell 7	Mean	SD
Perfusates 0 -24 h	1.30	0.45	0.74	2.60	0.75	1.08	1.15	0.77
Remaining Dose
Cell wash	3.11	1.88	2.26	2.16	1.78	2.33	2.25	0.47
Rinse	83.10	68.17	46.79	73.91	162.02	92.54	87.76	39.53
Skin Membrane	2.44	0.13	3.84	10.11	3.45	4.26	4.04	3.32
Subtotal	88.65	70.18	52.89	86.18	167.25	99.13	94.05	39.34
Recovery	89.96	70.63	53.63	88.78	168.00	100.21	95.20	39.31

Table 5. Recovery of radioactivity following application of [14C]-test substance to rat skin membrane (Group Q1, dose level A2)

Dose applied	[% of Dose] 455 µg/cm2
Cell No.	Cell 2	Cell 3	Cell 4	Cell 5	Cell 6	Cell 7	Mean	SD
Perfusates 0 -24 h	8.55	3.16	8.66	2.59	6.81	8.52	6.38	2.81
Remaining dose
Cellwash	1.96	3.15	1.25	3.21	0.69	1.40	1.94	1.04
Rinse	76.53	76.20	80.45	80.79	85.77	81.82	80.26	3.56
Skin Membrane	13.80	18.56	10.44	12.65	6.18	8.17	11.63	4.40
Subtotal	92.30	97.91	92.14	96.66	92.64	91.39	93.84	2.73
Recovery	100.85	101.07	100.79	99.24	99.45	99.91	100.22	0.79

Table 6. Recovery of radioactivity following application of [14C]-test substance to human skin membrane (Group Q2, dose level A1) in % dose

Cell No	Cell 8	Cell 9	Cell 10	Cell 11	Cell 12	Cell 13	Cell 14	Mean	SD
Perfusates 0-24h	0.91	0.67	0.46	0.53	0.49	0.99	0.76	0.96	0.21
Remaining dose
Cellwash	5.03	6.36	1.47	1.74	1.40	3.04	2.46	3.07	1.92
Rinse	114.63	100.16	81.64	126.79	55.53	47.87	84.44	87.58	29.55
Skin Membrane	2.92	12.91	0.30	0.92	0.12	6.25	7.36	4.40	4.73
Subtotal	122.58	119.43	83.41	131.46	57.05	57.16	94.26	95.05	30.80
Recovery	123.48	120.10	83.88	131.99	57.54	58.15	95.02	95.74	30.79

Table 7. Recovery of radioactivity following application of [14C]-test substance to human skin membrane (Group Q2, dose level A2) in % dose

Cell No.	Cell 9	Cell 10	Cell 11	Cell 12	Cell 13	Cell 14	Mean	SD
Perfusates 0 -24 h	0.28	0.37	0.38	0.33	0.40	0.28	0.34	0.05
Remaining Dose
Cellwash	1.76	0.42	0.29	1.00	0.46	0.76	0.78	0.54
Rinse	93.40	97.95	98.94	94.72	96.71	96.56	96.38	2.04
Skin	4.07	1.56	1.13	3.62	1.76	2.32	2.41	1.18
Subtotal	99.23	99.93	100.36	99.33	98.93	99.63	99.57	0.52
Recovery	99.52	100.30	100.75	99.67	99.34	99.91	99.91	0.53

Calculation dermal absorption according to the dermal absorption EFSA guidance (2017). The values were calculated as follow: absorption (mean)+ SD*k.

Table 8. Calculations human skin membrane (Low dose):

	[% of Dose]
Dose applied	0.9 µg/cm2
Cell No	Cell 8	Cell 9	Cell 10	Cell 11	Cell 12	Cell 13	Cell 14	Mean	SD
Perfusates 0-24h	0.91	0.67	0.46	0.53	0.49	0.99	0.76	0.96	0.21
Remaining dose
Cellwash	5.03	6.36	1.47	1.74	1.40	3.04	2.46	3.07	1.92
Rinse	114.63	100.16	81.64	126.79	55.53	47.87	84.44	87.58	29.55
Skin Membrane	2.92	12.91	0.30	0.92	0.12	6.25	7.36	4.40	4.73
Subtotal	122.58	119.43	83.41	131.46	57.05	57.16	94.26	95.05	30.80
Recovery	123.48	120.10	83.88	131.99	57.54	58.15	95.02	95.74	30.79
Total: Perfusates+ Skin membrane	3.83	13.58	0.76	1.45	excluded	excluded	8.12

Absorption mean % = (3.83 +13.58 +0.76 +1.45 +8.12)/5 = 5.55 %

Std = 5.33

Multiplication factor (k) = 1.2

Dermal absorption% = 5.55 + (5.33*1.2) = 12.0 % (Cell 12 and cell 13 were excluded in the calculation of the mean absorption due to low recovery).

Table 9. Calculations human skin membrane (high dose)

	[% of Dose]
Dose applied	455 µg/cm2
Cell No.	Cell 9	Cell 10	Cell 11	Cell 12	Cell 13	Cell 14	Mean	SD
Perfusates 0 -24 h	0.28	0.37	0.38	0.33	0.40	0.28	0.34	0.05
Remaining Dose
Cellwash	1.76	0.42	0.29	1.00	0.46	0.76	0.78	0.54
Rinse	93.40	97.95	98.94	94.72	96.71	96.56	96.38	2.04
Skin	4.07	1.56	1.13	3.62	1.76	2.32	2.41	1.18
Subtotal	99.23	99.93	100.36	99.33	98.93	99.63	99.57	0.52
Recovery	99.52	100.30	100.75	99.67	99.34	99.91	99.91	0.53
Total: Perfusates + Skin membrane	4.35	1.93	1.51	3.95	2.16	2.60

Absorption mean % = (4.35 +1.93+ 1.51+ 3.95+ 2.16+ 2.60)/6 = 2.75%

Std: 1.15

Multiplication factor (k) = 1.00

Dermal absorption % = 2.75 + (1.15*1) = 3.9%

Rat skin membrane:

Low dose: Cell 3, 4 and 6 should be excluded because of too low or too high recovery. The number of remaining cells is too limited to draw a conclusion on absorption.

Table 10. Calculations rat skin membrane (high dose)

	[% of Dose]
Dose applied	455 µg/cm2
Cell No.	Cell 2	Cell 3	Cell 4	Cell 5	Cell 6	Cell 7	Mean	SD
Perfusates 0 -24 h	8.55	3.16	8.66	2.59	6.81	8.52	6.38	2.81
Remaining dose
Cellwash	1.96	3.15	1.25	3.21	0.69	1.40	1.94	1.04
Rinse	76.53	76.20	80.45	80.79	85.77	81.82	80.26	3.56
Skin Membrane	13.80	18.56	10.44	12.65	6.18	8.17	11.63	4.40
Subtotal	92.30	97.91	92.14	96.66	92.64	91.39	93.84	2.73
Recovery	100.85	101.07	100.79	99.24	99.45	99.91	100.22	0.79
Total: Perfusates + Skin membrane	22.35	21.72	19.10	15.24	12.99	16.69

Absorption mean % = (22.35 +21.72 +19.10+ 15.24+ 12.99 + 16.69) /6 = 18.02%

Std = 3.70

Multiplication factor = 1.00

Dermal absorption % = 18.02 + (3.70*1) = 21.7 %

Conclusions:

The study demonstrated the amount of the test substance absorbed through human skin membrane and rat skin membrane over 24 hour with 0.9 µg/cm2 (low dose) and 455 µg/cm2 (high dose). The recalculated values for sum of % receptor fluid + % skin sample + k(n=5 for low dose or n=6 for high dose) x SD were 12% and 3.90% for 0.9 µg/cm2 and 455 µg/cm2 of the test substance for human skin membrane. The recalculated value for rat skin membrane was 21.7% for 455 µg/cm2 of the test substance. No conclusion can be made for the low dose rat skin based on the obtained data due to the limited number of remaining cells.

Executive summary:

The percutaneous penetration of the test substance was determined in vitro using split thickness skin membranes from rat and human skin according to OECD TG 428 and GLP principles. The skin membranes were set up in flow-through diffusion cells, the formulated [14C] radiolabelled test substance applied onto the skin membranes and the perfusates collected at defined time intervals. Two dose levels were used: the low dose level Al reflects a typical concentration recommended for use, e.g. pome and stone fruits (5 -10 g a.i./100 L), while the high dose level A2 represents the concentration of active ingredient in the undiluted formulation (50 g a.i./L).

Results showed that within 24 hours the portion of radiolabelled test substance penetrating through rat skin membranes accounted for 1.2 % and 6.4 % of the low and high dose, respectively. The human skin membranes showed a significantly lower permeability to the test susbtance than the rat skin membranes. At the low dose level all determined values in the perfusates were below the limit of determination (LQ) and at the high dose level only 0.3 % of the dose penetrated through human skin membranes within 24 hours of exposure. A distinct species difference in respect to the penetration of the test substance is also reflected by the flux. The flux at steady-state conditions was determined to be 0.0009 and 1.707µg/cm2/h through rat skin membranes at the low and high dose level, respectively. For the human skin membranes the flux could be calculated only for the high dose level, accounting for 0.106 pg/cm2/h. For the low dose levelthe calculation of the flux was not applicable, since all values were below the limit of determination. Making a conservative assumption of the flux for the low dose level based on the LQ values (0.0004μg/cm2/h) the human/rat ratio of the flux was at least 1:2 for this low dose level. For the high dose level, for which the flux for both species was able to be more accurately determined, the human/rat ratio was about 1:16.

In conclusion, the cumulative penetration of the formulated [14C)- test substance through rat skin membrane at the low dose level Al (1:500 v/v dilution) was only 1.2 % of the applied dose during 24 hours of exposure. Human skin membrane exposed under the same conditions was less permeable. All determined values in the perfusates were at or below the limit of determination (LQ). Therefore, the differences between rat and human skin membranes could not be calculated. Making a conservative assumption of the flux for the low dose level based on the LQ values (0.0004 gg/cm2/h) the human/rat ratio was at least 1:2 for this low dose level. At the high dose level A2 (undiluted formulation) 6.4 % and 0.3 % of the applied dose penetrated within 24 hours through the rat and human skin membranes respectively. The flux was calculated to be 1.707 µg/cm2/h for rat skin membrane and 0.106 μg/cm2/h for human skin membrane. Thus, the flux ratio was 1:16 (human/rat). The difference between rat and human skin membranes is evident when the relative amounts of the test substance that have penetrated the skin membranes are compared. At both dose levels the test substance penetrated faster and to a higher extent through rat skin membranes compared to human skin membranes, more markedly at the high dose level. The study demonstrated the amount of the test substance absorbed through human skin membrane and rat skin membrane over 24 hour with 0.9 µg/cm2 (low dose) and 455 µg/cm2 (high dose). The study demonstrated the amount of the test substance absorbed through human skin membrane and rat skin membrane over 24 hour with 0.9 µg/cm2 (low dose) and 455 µg/cm2 (high dose). The recalculated values for sum of % receptor fluid + % skin sample + k(n=5 for low dose or n=6 for high dose) x SD were 12% and 3.90% for 0.9 µg/cm2 and 455 µg/cm2 of the test substance for human skin membrane. The recalculated value for rat skin membrane was 21.7% for 455 µg/cm2 of the test substance. No conclusion can be made for the low dose rat skin based on the obtained data due to the limited number of remaining cells.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Sep 1989 to 29 Aug 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Objective of study:

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

1984

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese MAFF

Version / remarks:

1985

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984

Qualifier:

equivalent or similar to guideline

Guideline:

other: 94/79/EC

Version / remarks:

1994

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

rat

Strain:

other: Tif: RAIf

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: 30% ethanol and 70% polyethylene glycol (PEG 200) (v/v)

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

0.5 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose / concentration:

The analysed samples were pooled.

Control animals:

no

Metabolites identified:

yes

Details on metabolites:

The test substance was poorly metabolised by male and female rats after a single oral dose of 100 mg/kg. Spectroscopic analyses showed that the major metabolite in faeces, fat, and probably also in the other tissues was the unchanged test substance. Only about 1% of an oral dose of the test substance was metabolised by deacylation followed by cleavage of the ureido group. Two further similarly minor metabolites were characterised by chromatographic comparison with the authentic reference compounds.

The faecal pools for male and female rats were almost quantitatively extracted with methanol. The TLC pattern of the combined methanol extracts revealed one major metabolite fraction designated F3. This

accounted for 98 % of faecal radioactivity, corresponding to 75% of the dose. High voltage electrophoresis of the combined methanol extracts revealed the presence of minor amounts of acidic metabolites, representing less than 0.5 % of dose. The acidic metabolites were identified as M4 and M6 by cochromatography with authentic reference compounds. No alkaline metabolites were detected as no radioactivity moved towards the cathode.

Table 1. Quantitative metabolite profile in 8-48 hour faecal extract pool from male and female rats and assignment of metabolites (% of dose).

Metabolite Fraction	% of dose	Assignment of metabolites
F1	0.5	Metabolite 2F
F2	0.4	M4
F3	74.8	Test substance
F4-6	0.3	M6
Total	76.0	-
Actual Recovery	99%	-

* represents only a part of the metabolite fraction

After further purification of the methanol extract by LC and HPLC, metabolite F2 was isolated. Due to the small amount available no 1H-NMR spectrum could be obtained. However, mass spectroscopic analysis revealed the presence of chlorine. Fragment ions at m/e 327 and 176 were observed, but no molecular ion species could be established. The fragmentation pattern indicated that the metabolite did not contain the 2,6- difluoro-benzoyl-moiety. Organic solvent extraction of the single pool of fat from male and female rats extracted approximately 93% of the radioactivity present. Purification by preparative TLC yielded two fractions. The fraction containing almost all the radioactivity was further purified by HPLC. It was shown by mass spectroscopy and by cochromatography to be identical to the parent compound lufenuron. By chromatographic comparison the second fraction was corresponded to Metabolite F2.

Mostly unchanged lufenuron was excreted in faeces and was also retained in tissues, predominantly in fat, demonstrating that the test substance was poorly metabolised. It is assumed that a minor route of degradation is cleavage of the benzamide-moiety yielding the urea M4 and 2,6-difluorobenzoic acid. Further cleavage of the ureido moiety of M4 leads to the aniline M6. It is established that rats excrete 2,6-difluorobenzoic acid largely unchanged.

Conclusions:

The test substance was poorly metabolised by male and female rats after a single oral dose of 100 mg/kg. Spectroscopic analyses showed that the major metabolite in faeces, fat, and probably also in the other tissues was the unchanged test substance. Only about 1% of an oral dose of the test substance was metabolised by deacylation followed by cleavage of the ureido group. Two further similarly minor metabolites were characterised by chromatographic comparison with the authentic reference compounds, M4 and M6

Executive summary:

The test substance was administered to young, adult male and female Tif: RAIf (SPF) rats in various groups according to OECD TG 417 and GLP principles. The test substance was administered in a mixture of 30% ethanol and 70% polyethylene glycol (PEG 200) (v/v). Faeces and fat collected from rats administered a single oral dose of 100 mg [14C]-test substance/kg were used in the present study. Urinary metabolites were not analysed because they represented less than 1% of the dose. Collectively, the faecal pools analysed represented 76% of the dose and the fat, as the predominant tissue residue, approximately 11%.

Results showed, that the faecal pools for male and female rats were almost quantitatively extracted with methanol. The TLC pattern of the combined methanol extracts revealed one major metabolite fraction designated F3. This accounted for 98% of faecal radioactivity, corresponding to 75% of the dose. High voltage electrophoresis of the combined methanol extracts revealed the presence of minor amounts of acidic metabolites, representing less than 0.5% of dose. The acidic metabolites were identified as M4 and M6 by co chromatography with authentic reference compounds. No alkaline metabolites were detected as no radioactivity moved towards the cathode. Mostly unchanged test substance was excreted in faeces and was also retained in tissues, predominantly in fat, demonstrating that lufenuron was poorly metabolised. It is assumed that a minor route of degradation is cleavage of the benzamide-moiety yielding the urea M4 and 2,6-difluorobenzoic acid. Further cleavage of the ureido moiety of M4 leads to the aniline M6. It is established that rats excrete 2,6-difluorobenzoic acid largely unchanged.

In conclusion, the test substance was poorly metabolised by male and female rats after a single oral dose of 100 mg/kg. Spectroscopic analyses showed that the major metabolite in faeces, fat, and probably also in the other tissues was the unchanged test substance. Only about 1% of an oral dose of the test substance was metabolised by deacylation followed by cleavage of the ureido group. Two further similarly minor metabolites were characterised by chromatographic comparison with the authentic reference compounds, M4 and M6

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 Jun 2002 to 6 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

excretion

metabolism

tissue distribution

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Qualifier:

equivalent or similar to guideline

Guideline:

other: 94/79/EC

Version / remarks:

1994

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

1995

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese MAFF

Version / remarks:

1985

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Species:

rat

Strain:

other: HanBrl: WIST (SPF)

Sex:

male

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

200:ethanol (7:3 v/v)

Duration and frequency of treatment / exposure:

Once daily for up to 14 days starting on Day 0. Four subgroups of 4 rats were sacrificed on Day 1, 7, 14 and 20.

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

Representing doses of 0.48 - 0.65 mg [14C]-test substance/kg (0.86 -1.2 MBq/kg)

No. of animals per sex per dose / concentration:

16

Control animals:

no

Details on absorption:

During the dosing phase there was a progressive increase in blood concentrations to maximal values of approximately 0.18 ppm equivalents test substance/g. Concentrations then declined gradually with an estimated elimination half-life of 9 days

Details on distribution in tissues:

The highest residues were present in fat (29 ppm test substance equivalents). Much lower and progressively smaller residue levels were present in adrenals (4.2 ppm), pancreas (3.2 ppm), and thyroid (3.0 ppm) liver (2.1 ppm), kidneys (1.3 ppm), heart (1.2 ppm), lungs (1.1 ppm), and thymus (1.1 ppm). All other tissues attained maximum levels below 1 ppm lufenuron equivalent. The calculated half-life (t½) for the depuration of tissue [14C]-residues, assuming a monophasic first order kinetics, typically ranged from 7 to 12 days. A faster depletion was observed for thyroid (t½ = 4 days). Elimination half-lives of residues from testes, lungs and fat ranged from 14 to16 days, although testes showed lower residues after 14 days than after 7 days during the accumulation phase. Seven days after the last of 14 consecutive daily doses, tissue residues represented approximately 38 % of the total administered radioactivity

Details on excretion:

By 7 days after the final dose, over 58% of the cumulative dose had been excreted. Urinary excretion accounted for only circa 1% of the total dose whilst faecal excretion accounted for the remainder. The un-excreted dose was accumulated in tissues and the rate of faecal excretion suggested that such tissue residues would be eliminated slowly and predominantly in faeces.

Metabolites identified:

yes

Details on metabolites:

Chromatographic analysis of urine revealed a metabolite profile, comprising at least 10 metabolite fractions. Although the total amount of radiolabelled metabolites was very low, based on very low urinary excretion, the patterns were qualitatively and quantitatively similar after single and repeated dosing.

Blood [14C]-concentrations during and following 14 daily doses are presented below. During the dosing phase there was a progressive increase in blood concentrations to maximal values of approximately 0.18 ppm equivalents test substance/g. Concentrations then declined gradually with an estimated elimination half-life of 9 days.

Table 1. Concentrations of radioactivity in blood during and following 14 daily oral doses of 0.5 mg [14C]-test substance/kg. (Values are expressed as ppm equivalents test substance/g of blood).

Day	ppm equiv/g	Day	ppm equiv/g	Day	ppm equiv/g
1	0.0319	8	0.1367	15	0.1569
2	0.0540	9	0.1574	16	0.1452
3	0.0812	10	0.1671	17	0.1295
4	0.0949	11	0.1702	18	0.1248
5	0.1071	12	0.1816	19	0.1145
6	0.1326	13	0.1767	20	0.1096
7	0.1359	14	0.1711

Excretion results during and following the 14 consecutive daily oral doses are tabulated below. Values are expressed both as percentages of each daily dose and of the total administered dose. By 7 days after the final dose, over 58% of the cumulative dose had been excreted. Urinary excretion accounted for only circa 1% of the total dose whilst faecal excretion accounted for the remainder. The un-excreted dose was accumulated in tissues and the rate of faecal excretion suggested that such tissue residues would be eliminated slowly and predominantly in faeces.

Table 2. Excretion of radioactivity during and following 14 daily oral doses of 0.5 mg [14C] - test substance/kg

		Urine		Faeces
Day of study		% of daily dose	% of total dose	% of daily dose	% of total dose
0 -	1 d	0.51	0.04	27.78	1.96
1 -	2 d	0.77	0.06	30.73	2.22
2 -	3 d	0.70	0.05	32.97	2.37
3 -	4 d	0.77	0.06	43.23	3.22
4 -	5 d	0.77	0.06	43.52	3.12
5 -	6 d	0.76	0.05	46.54	3.32
6 -	7 d	0.77	0.05	46.15	3.27
7 -	8 d	0.81	0.06	49.82	3.49
8 -	9 d	0.94	0.07	45.67	3.22
9 -	10 d	0.86	0.06	51.15	3.70
10 -	11 d	1.16	0.08	47.82	3.39
11 -	12 d	1.05	0.08	52.25	3.74
12 -	13 d	1.04	0.07	55.51	3.91
13 -	14 d	1.11	0.08	55.64	3.96
0 - 14	Subtotal	-	0.86	-	44.62
14 -	15 d	0.93	0.07	39.28	2.80
15 -	16 d	0.71	0.05	33.47	2.38
16 -	17 d	0.66	0.05	34.31	2.44
17 -	18 d	0.69	0.05	30.07	2.14
18 -	19 d	0.67	0.05	29.72	2.12
19 -	20 d	0.55	0.04	26.63	1.90
Subtotal		-	1.18	-	58.39

- Not applicable

One rat in group T4 died on day 13 after a mis-dose

Tissue distribution results during and following the 14 consecutive daily oral doses are tabulated below. With the single exception of testes, all tissue concentrations of radioactivity increased to a maximum one day after the final dose. The results suggest that most tissue concentrations would plateau within 2 to 3 weeks of similar repeated dosing. The highest residues were present in fat (29 ppm lufenuron equivalents). Much lower and progressively smaller residue levels were present in adrenals (4.2 ppm), pancreas (3.2 ppm), and thyroid (3.0 ppm) liver (2.1 ppm), kidneys (1.3 ppm), heart (1.2 ppm), lungs (1.1 ppm), and thymus (1.1 ppm). All other tissues attained maximum levels below 1 ppm lufenuron equivalent. The calculated half-life (t½) for the depuration of tissue [14C]-residues, assuming a monophasic first order kinetics, typically ranged from 7 to 12 days. A faster depletion was observed for thyroid (t½ = 4 days). Elimination half-lives of residues from testes, lungs and fat ranged from 14 to16 days, although testes showed lower residues after 14 days than after 7 days during the accumulation phase. Seven days after the last of 14 consecutive daily doses, tissue residues

represented approximately 38 % of the total administered radioactivity. There was a similar distribution pattern between these profiles and those seen 7 days after a similar single oral dose (See table 3), although there was an approximate 10-fold difference in the comparative tissue concentrations.

Table 3. Concentrations of radioactivity in tissues during and following 14 daily oral doses of 0.5 mg [14C]-test substance/kg. (Values are expressed as μg equivs test substance/g of tissue)

Dosing	Multiple doses					Single
Necropsy	Residues [ppm test substance equivalents]				Half-life [days] t½	ppm equivs
Days after start of dosing Days after dosing	1 1	7 1	14 1	20 7		7 7
Adrenals	0.7418	2.3793	4.1879	2.3915	7	n.a.
Blood	0.0312	0.1105	0.1656	0.0991	8	0.0084
Bone	0.0957	0.3021	0.3297	0.2351	12	0.0389
Brain	0.0313	0.1113	0.1396	0.0816	8	0.0131
Fat	3.4809	21.1502	29.2452	22.6602	16	1.9142
Heart	0.2614	0.9261	1.2472	0.7746	9	0.0802
Kidneys	0.2923	1.0677	1.3481	0.8845	10	0.0879
Liver	0.4620	1.6005	2.1192	1.3460	9	0.1292
Lungs	0.2994	0.8266	1.0963	0.8338	15	0.0942
Muscle	0.1564	0.5764	0.6367	0.3956	9	0.0404
Pancreas	0.5957	2.1645	3.1696	2.1651	11	n.a.
Plasma	0.0395	0.1389	0.2317	0.1307	7	0.0104
Spleen	0.1600	0.5477	0.7242	0.5126	12	0.0465
Testes	0.0822	0.3673	0.2790	0.2076	14	0.0260
Thymus	0.1734	0.7284	1.0870	0.6185	7	0.0560
Thyroids	0.4131	2.4412	3.0236	1.0975	4	0.2200

n.a. – not applicable

Recovery of administered radioactivity

The mean recovery of total administered radioactivity to rats in group T4 is tabulated below. Approximately 1% of the dose was excreted in urine and 58% in faeces. Approximately 38% of the cumulative dose remained in tissues and the residual carcass at the conclusion of this study. The total recovery of administered radioactivity was greater than 97%. Based upon the analyses of tissues 1 day after a single oral dose, the presence of an estimated value of circa 80% of the dose in tissues and the residual carcass indicates that absorption of a 0.5 mg/kg oral dose was circa 80%.

Table 4. Recovery of radioactivity 7 days after 14 consecutive daily oral administrations of 0.5 mg [14C]-test substance/kg. (Values are expressed as percentages of total administered radioactivity)

Sample	% of dose
Urine	1.18
Faeces	58.39
Cage Wash	0.08
Total Excretion	59.65
Tissue residues	3.98
Carcass residues	33.60
Subtotal	37.58
Total Recovery	97.23

Metabolite analyses in urine and faeces

The results of solvent extraction are tabulated below. The efficiency of solvent extraction of radioactivity from urine was almost quantitative and from faecal pools ranged from 67-81%.

Table 5. Results of solvent extraction of urine and faeces

Urine	Percentage of pooled urine			Percentage of daily [14C]-dose
Pool (days)	Total	Extract 1	Extract 2	Total	Extract 1	Extract 2
0 – 1	100.0	7.3	92.7	0.5	0.04	0.46
6 – 7	100.0	13.9	86.0	0.8	0.11	0.69
13 – 14	100.0	15.4	84.6	1.1	0.17	0.93
Faeces	Percentage of pooled faeces			Percentage of daily [14C]-dose
Pool (days)	Total	Extracted	Non- extractable	Total	Extracted	Non- extractable
0 – 1	100.0	80.6	19.4	27.8	22.4	5.4
6 – 7	100.0	75.6	24.4	46.2	34.9	11.3
13 – 14	100.0	67.3	32.7	55.6	37.4	18.2

Chromatographic analysis of urine revealed a metabolite profile, comprising at least 10 metabolite fractions. Although the total amount of radiolabelled metabolites was very low, based on very low urinary excretion, the patterns were qualitatively and quantitatively similar after single and repeated dosing.

Table 6. Urinary metabolite profile

Urinary metabolite pattern (% of daily dose)
Sampling Time	Day 0-1	Day 6-7	Day 13-14
Metabolite Fraction
1	0.02	0.04	0.05
2	0.07	0.07	0.07
3	0.01	0.03	0.04
4	0.04	0.11	0.21
5	0.09	0.14	0.25
6	0.02	0.06	0.04
7	0.14	0.09	0.11
8	0.01	0.02	0.03
9	0.01	0.05	0.07
10	0.06	0.09	0.09
Extraction 2 Extraction 1	0.46 0.04	0.69 0.11	0.93 0.17
Total	0.50	0.80	1.10

TLC analysis of the faecal extracts revealed at least 4 metabolite fractions. The extractability and the metabolite pattern of the faeces were essentially identical for all three selected sampling intervals with slight quantitative variations mainly between the first time interval (0-1 day) and the later selected time intervals. The major metabolite fraction (3) corresponds to unchanged lufenuron, accounting for circa 30 % of the daily dose. Apart from unchanged parent, only minor metabolites were present in the faecal extracts.

Table 7. Faecal metabolite profile

Sampling Time	Faecal metabolite pattern (% of daily dose)
	Day 0-1	Day 6-7	Day 13-14	Assignment
Metabolite Fraction				Test substance
1	1.0	1.7	2.4
2	0.6	1.2	1.3
3	20.5	29.6	32.3
4	0.3	2.4	1.5
Extracted Non-extractable	22.4 5.4	34.9 11.3	37.4 18.2
Total	27.8	46.2	55.6

Conclusions:

Repeated daily oral doses of 0.5 mg [14C]-test substance/kg were rapidly and extensively absorbed into systemic circulation. Blood concentrations increased to a plateau after 11 daily doses and most tissue concentrations approached a plateau after 14 daily doses. The highest tissue residues were present in fat with much lower concentrations in other tissues. The half-life of elimination of tissue [14C]-residues ranged from 4 to 16 days and by 7 days after the cessation of dosing, 38% of the total dose remained in tissues. Approximately 1% and 50% of each daily dose were excreted in urine and faeces, respectively and the predominant component extracted from faeces was the unchanged test substance. Metabolite profiles after 1, 7 and 14 doses were essentially identical. Hence, the rates and routes of excretion and tissue distribution patterns were not affected by repeated daily dosing, compared to a single oral dose.

Executive summary:

The absorption and excretion was investigated in 16 male HanBrl: WIST rats by oral gavage of the test substance according to OECD TG 417 and GLP principles. The dosing was performed by 14 consecutive daily doses at a nominal dose level of 0.5 mg/kg body weight. Four animals were assigned to each of four subgroups (T1-T4) which were sacrificed at 4 different time points after the start of administration and the test substance related residues were determined in tissues and organs. The excreted radioactivity was determined in urine and faeces of subgroup T4 at daily intervals. At termination, the following tissues were collected: adrenals, bone, brain, fat (abdominal), heart, kidneys, liver, lungs, muscle (skeletal), pancreas, plasma, spleen, testes, thymus, thyroid, whole blood and residual carcass. Plasma was taken for direct liquid scintillation counting (LSC) together with urine and cage wash samples. Other tissue samples were prepared for LSC by solubilisation or by sample oxidation together with faecal samples. Representative urine pools were prepared from samples collected over days 0-1, 6-7 and 13- 14. Representative pools of faeces collected over days 0-1, 6-7 and 13-14 were serially extracted with acetonitrile and acetonitrile: water (4:1 v/v). For each time interval, the extracts were combined and concentrated for TLC analysis.

Results showed, that the test substance was rapidly and almost completely absorbed from the gastrointestinal tract into the systemic circulation. The rate and extent of excretion was moderate. A steady state in terms of excretion was reached 6 days after start of dosing. About 50 % of the daily dose were excreted via faeces whereas only 1 % of the daily dose was excreted with the urine. After the dosing period the daily excretion decreased very slowly. In sum only 58 % and 1 % of the total administered dose was excreted with the faeces and the urine, respectively. In a previous study the extent of absorption after single oral administration at the low dose level (0.5 mg/kg body weight) was estimated to be 45 % of the dose. The results of the present study indicate a significantly higher extent of absorption. The tissue residues, determined 24 hours after the first administration, accounted in sum for about 80 % of the dose which has to be considered as absorbed from the gastrointestinal tract. The blood kinetics showed increasing concentrations of radioactivity with ongoing administrations up to 11 days after the start of dosing. Thereafter the concentration in blood remained almost constant at a plateau level of about 0.17 ppm test substance equivalents during the further dosing period. After the last of 14 consecutive daily doses the residues in whole blood decreased very slowly not reaching half the maximum concentration within 7 days. The tissue residues were determined at 4 different time points during and after the dosing period, i.e. 1, 7, 14, and 20 days. The residues in all selected tissues and organs increased with ongoing administrations reaching the maximum residue level 1 day the last dose. The highest residue level was found in fat accounting for 29 ppm test substance equivalents. Lower but also remarkable residue levels were determined in adrenals (4.2 ppm), pancreas (3.2 ppm), and thyroids (3.0 ppm). The determined maximum residues levels in liver, kidneys, heart, lungs, and thymus were in the range of 1-2 ppm. All other tissues and organs reached maximum residue levels not exceeding 1 ppm test substance equivalents. Seven days after the last of 14 consecutive daily doses about 38 % of the total administered dose were still present in tissues and organs. The calculated half-lives (t_1/2) for the depuration of the residual radioactivity from tissues and organs, assuming a monophasic first order kinetics, were for most of the selected tissues in the range of 7 to 12 days. A somewhat higher depletion rate was calculated for thyroids (t_1/2=4 days). A higher persistence of the residues was found in testes, lungs and fat, i.e. (t_1/2) =14-16 days). The tissue residues 7 days after multiple dosing were about 10 fold higher as compared to a single oral administration of the test substance. The metabolite pattern of urine and faeces, investigated at three different time intervals during the dosing period, i.e. day 0-1, day 6-7, and day 13-14, were not influenced by multiple dosing

In conclusion, repeated daily oral doses of 0.5 mg [14C]-test substance/kg were rapidly and extensively absorbed into systemic circulation. Blood concentrations increased to a plateau after 11 daily doses and most tissue concentrations approached a plateau after 14 daily doses. The highest tissue residues were present in fat with much lower concentrations in other tissues. The half-life of elimination of tissue [14C]-residues ranged from 4 to 16 days and by 7 days after the cessation of dosing, 38% of the total dose remained in tissues. Approximately 1% and 50% of each daily dose were excreted in urine and faeces, respectively and the predominant component extracted from faeces was the unchanged test substance. Metabolite profiles after 1, 7 and 14 doses were essentially identical. Hence, the rates and routes of excretion and tissue distribution patterns were not affected by repeated daily dosing, compared to a single oral dose.

Description of key information

- The test substance was rapidly absorbed from the gastrointestinal tract.

- Oral absorption: approximately 70% based on comparison of AUC (0-120h) of single oral and intravenous dose; OECD TG 417; Booth; 2004

- Metabolism: the test substance was poorly metabolised, with only about 1 % of an oral dose being metabolised by deacylation followed by cleavage of the ureido group, OECD TG 417; Thanei, 1990

- Excretion: the rates and routes of excretion and tissue distribution patterns were not affected by repeated daily dosing, compared to a single oral dose, OECD TG 417; Hassler 2003

- Dermal absorption: 12% (diluted product), in vitro absorption study in human skin, OECD TG 428; Hassler 2003

- Inhalation absorption: Based on the lack of information on inhalation absorption, the default absorption value from the REACH guidance (Chapter 8, R.8.4.2) is used for DNEL derivation, namely: 100%

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

70

Absorption rate - dermal (%):

12

Absorption rate - inhalation (%):

100

Additional information

Absorption: 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 (Booth 2004)

Dermal route: The study demonstrated the amount of the test substance absorbed through human skin membrane and rat skin membrane over 24 hour with 0.9 µg/cm2 (low dose) and 455 µg/cm2 (high dose). The recalculated values for sum of % receptor fluid + % skin sample + k(n=5 for low dose or n=6 for high dose) x SD were 12 % and 3.9 % for 0.9 µg/cm2 and 455 µg/cm2 of the test substance for human skin membrane. The recalculated value for rat skin membrane was 21.7% for 455 µg/cm2 of the test substance. No conclusion can be made for the low dose rat skin based on the obtained data due to the limited number of remaining cells (Hassler, 2003) according to the dermal absorption EFSA guidance (2017).

Distribution: Distributed mainly to the fat and in smaller amounts in order organs (Bissig, 1990)

Metabolism: There was no evidence that the test substance or its metabolites were distributed into particular organs or brain areas (Okada, 1998). Only about 1% of an oral dose of the test substance was metabolised by deacylation followed by cleavage of the ureido group. Two further similarly minor metabolites were characterised by chromatographic comparison with the authentic reference compounds (Thanei, 1990). In another study, the faecal metabolite pattern was independent of sex and pre-treatment and showed slightly dose-dependent differences. As a consequence of the higher amount excreted with the faeces by the high dosed animals, the major metabolite fraction present in the faeces extracts covered more percent of the administered dose in the faeces of the 100 mg/kg bw/day group (about 80%) than in the faeces of the 0.5 mg/kg bw/day group (about 40%). This metabolite fraction corresponded to unchanged test substance. Most of the biliary metabolite fractions were more polar than the faecal metabolites. One metabolite fraction (0.1% of dose) was characterised as unchanged test substance. Another metabolite fraction (0.1% of dose) co-chromatographed with the authentic reference compound M1. A further fraction behaved like the free amine M2 (Bissig, 1990).

Excretion: The major route of elimination is the faeces, 50% after a low oral dose of 0.5 mg/kg and 80% after a high oral dose of 100 mg/kg. Ca 67% after i.v. administration. Less than 1% was excreted in the urine. All species investigated showed a slow elimination. Depletion of tissue residues followed bi-phasic first order kinetics with half-life times of 2-6 days for phase 1 and up to 37 days in phase 2. The excretion rate was highly dose-dependent but only slightly influenced by the sex of the animals (Bissig, 1990).