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

Dermal absorption

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

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:

other: Rat and human skin membrane

Sex:

not specified

Administration / exposure

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.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Percutaneous absorption

Percutaneous absorption 1

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)

Percutaneous absorption 2

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)

Percutaneous absorption 3

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)

Percutaneous absorption 4

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)

Any other information on results incl. tables

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 %

Applicant's summary and conclusion

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.