Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Mar 2008 to 18 Apr 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Radiolabelling:

yes

Remarks:

[pyrazolyl-5-14C]

Test animals

Species:

other: Human skin membrane

Administration / exposure

Type of coverage:

open

Vehicle:

water

Doses:

Concentrate (125 g/L) and two dilutions (nominally 1/83 and 1/1250)

Results and discussion

Percutaneous absorption

Percutaneous absorption 1

Time point:

24 h

Concentrate / Dilution:

concentrate

Absorption:

2 %

Remarks on result:

other: dermal absorption = (stratum corneum + remaining epidermis + receptor fluid) + k * SD

Percutaneous absorption 2

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

1/83 v/v

Absorption:

6.1 %

Remarks on result:

other: dermal absorption = (stratum corneum + remaining epidermis + receptor fluid) + k * SD

Percutaneous absorption 3

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

1/1250 v/v

Absorption:

19.13 %

Remarks on result:

other: dermal absorption = (stratum corneum + remaining epidermis + receptor fluid) + k * SD

Any other information on results incl. tables

TEST SUBSTANCE ABSORPTION RATE

- Formulation concentrate: The absorption rate between 0 - 8 hours was 0.037 µg/cm2/h, after which it increased to 0.066 µg/cm2/h between 8 - 16 hours. Fastest absorption occurred between 16 - 24 hours, when test substance was absorbed at a rate of 0.085 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.061 µg/cm2/h.

-1/83 v/v aqueous spray dilution;The absorption rate between 0 - 6 hours the absorption rate was 0.008 µg/cm2/h, after which it increased to 0.016 µg/cm2/h between 6 - 12 hours. Fastest absorption occurred between 12- 24 hours, when test substance was absorbed at a rate of 0.018 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.015 µg/cm2/h.

-/1250 v/v aqueous spray dilution:test substance absorption was essentially linear throughout the entire 24 hour exposure period. Between 0-24 hours the mean absorption rate was 0.003 µg/cm2/h.

 

MASS BALANCE AND TEST SUBSTANCE DISTRIBUTION

-Formulation concentrate: Mean recovery of the applied test material was 104 %. The majority of the applied dose, 97.5 % was removed by gentle skin washing 24 hours after application. The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 0.122 %. This percentage equated to 1.48 µg/cm2. A total of 1.43 % of the applied dose remained in the epidermal membrane following a 24 hour skin washing procedure. Of this total, 0.261 % was present in the outer layers of the stratum corneum.

- 1/83 v/v aqueous spray dilution: Mean recovery of the applied test material was 101 %. Skin washing 24 hours after application removed 95.5 % of the applied dose. The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 2.66 %. In terms of actual amounts this percentage equated to 0.360 μg/cm2. A total of 1.19 % of the applied dose remained in the epidermal membrane following a 24 hour skin washing procedure. Of this total, 0.470 % was present in the outer layers of the stratum corneum.

- 1/1250 v/v aqueous spray dilution: Mean recovery of test material was 104 % of the applied dose. Skin washing 24 hours after application removed 94.2 % of the applied dose. The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 6.19 %. In terms of actual amounts this percentage equated to 0.063 μg/cm2. A total of 2.90 % of the applied dose remained in the epidermal membrane following a 24 hour skin washing procedure. Of this total, 0.761 % was present in the outer layers of the stratum corneum.

Table 1 Summary of Test substance Absorption through Human Epidermis

Application of Test Materials	Mean Absorption Rates		Mean Amount and Percentage of Dose Absorbed
	Time period (h)	Absorption rate (mg/cm^2/h ±SEM)	Time (h)	Amount (mg/cm^2)	Percentage absorbed
Formulation concentrate (122 g substance / L)	0-8	0.037 ± 0.007	6	0.191	0.016
10 mg/cm2 (1218μg/cm2)	8-16	0.066 ± 0.013	8	0.283	0.023
Unoccluded	16-24	*0.085 ± 0.018	10	0.387	0.032
Duration of exposure: 24 h	0-24	0.061 ± 0.012	24	1.48	0.122
n = 6			LOQ	0.038	0.003
1/83 v/v aqueous spray dilution (1.35 g substance / L)	0-6	0.008 ± 0.001	6	0.047	0.346
10 mg/cm^2(13.5 mg ai/cm^2) Unoccluded	6-12 12-24	0.016 ± 0.004 *0.018 ± 0.005	8 10	0.075 0.107	0.558 0.792
Duration of exposure: 24 h	0-24	0.015 ± 0.004	24	0.360	2.66
n = 6			LOQ	0.0004	0.003
1/1250 v/v aqueous spray dilution.
(0.10 g substance / L)			6	0.015	1.43
10 mg/cm^2(1.02 mg ai/cm^2)	0-24	*0.003 ± 0.001	8	0.020	1.95
Unoccluded			10	0.025	2.48
Duration of exposure: 24 h			24	0.063	6.19
n = 6			LOQ	0.0002	0.024

*Fastest rate of absorption over the 24h time course.

Table 2 Summary of Test Substance Distribution in the Test System after 24 Hours

Formulation concentrate
Test Compartment n = 5	μg test substance per cm²		% of applied dose
	Mean	SEM	Mean	SEM
Donor chamber	65.2	8.54	5.36	0.701
Skin wash	1187	18.5	97.5	1.522
Stratum corneum	3.18	0.356	0.261	0.029
Remaining epidermis	14.2	2.64	1.17	0.216
*Absorbed	1.48	0.298	0.122	0.024
Total recovered	1272	21.0	104	1.72
1/83 v/v dilution
Test Compartment n = 6	μg test substance per cm²		% of applied dose
	Mean	SEM	Mean	SEM
Donor chamber	0.212	0.145	1.57	1.07
Skin wash	12.9	0.288	95.5	2.13
Stratum corneum	0.064	0.021	0.470	0.156
Remaining epidermis	0.098	0.034	0.723	0.254
*Absorbed	0.360	0.096	2.66	0.708
Total recovered	13.7	0.370	101	2.73
1/1250 v/v dilution
Test Compartment n = 6	μg test substance per cm²		% of applied dose
	Mean	SEM	Mean	SEM
Donor chamber	0.004	0.002	0.358	0.176
Skin wash	0.959	0.052	94.2	5.12
Stratum corneum	0.008	0.002	0.761	0.185
Remaining epidermis	0.022	0.005	2.14	0.488
*Absorbed	0.063	0.026	6.19	2.55
Total recovered	1.06	0.026	104	2.55

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Table 3 Calculations human skin membrane (Concentrate) - Individual Distribution of test substance from the Formulation Concentrate in the Test System

Test Compartment	Percentage of Dose Recovered (%): concentrate
	Cell 50	Cell 51	Cell 52	Cell 53	Cell 54	Cell 56
Donor chamber	3.73	7.40	6.43	6.69	3.25	4.63
Skin wash	91.6	101	94.5	98.8	101	98.0
Stratum corneum	0.18	0.17	0.334	0.308	0.263	0.313
Remaining epidermis	1.64	1.71	1.48	0.363	0.836	0.981
Absorbed	0.126	0.068	0.041	0.179	0.121	0.195
Total recovered	97.3	110	103	106	106	104

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Absorption mean % = (1.95 + 1.95 + 1.86 + 0.85 + 1.22 + 1.49)/6 = 1.55 %

Std = 0.45

Multiplication factor (k) = 1

Dermal absorption% = 1.55 + (0.45*1) = 2.00 %

Table 4 Calculations human skin membrane (1/83 v/v Spray) - Individual Distribution of test substance from the 1/83 v/v Spray Strength Dilution in the Test System

Test Compartment	Percentage of Dose Recovered (%): 1/83 v/v Spray
	Cell 90a	Cell 91a	Cell 92a	Cell 93a	Cell 94a	Cell 95a
Donor chamber	6.71	0.065	0.739	1.87	0.005	0.004
Skin wash	89.6	91.2	96.1	102	101	92.8
Stratum corneum	1.10	0.181	0.478	0.722	0.165	0.173
Remaining epidermis	1.53	0.192	0.594	1.48	0.310	0.235
Absorbed	4.26	1.40	3.37	4.90	1.17	0.885
Total recovered	103	93.1	101	111*	103	94.1

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Absorption mean % = (6.89 + 1.77 + 4.44 + 1.65 + 1.29)/5 = 3.21 %

Std = 2.41

Multiplication factor (k) = 1.2

Dermal absorption% = 3.21 + (2.41*1.2) = 6.10 %

*(Cell 93a were excluded in the calculation of the mean absorption due to high recovery).

Table 5 Calculations human skin membrane (1/1250 v/v Spray) - Individual Distribution of test substance from the 1/1250 v/v Spray Strength Dilution in the Test System

Test Compartment	Percentage of Dose Recovered (%): 1/1250 v/v Spray
	Cell 35b	Cell 36b	Cell 37b	Cell 42b	Cell 44b	Cell 46b
Donor chamber	<0.001	<0.001	0.898	0.385	<0.001	0.866
Skin wash	104	106	84.8	101	94.8	74.1
Stratum corneum	0.112	0.559	0.816	0.892	0.691	1.50
Remaining epidermis	0.863	1.52	4.21	1.65	1.77	2.82
Absorbed	1.48	2.20	4.50	1.88	9.82	17.3
Total recovered	107	111*	95.2	105	107	96.5

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Absorption mean % = (2.46 + 9.53 + 4.42 + 12.28 + 21.62)/5 = 10.06 %

Std = 7.56

Multiplication factor (k) = 1.2

Dermal absorption% = 10.06 + (7.56*1.2) = 19.13 %

* (Cell 36b were excluded in the calculation of the mean absorption due to high recovery).

Applicant's summary and conclusion

Conclusions:

Based on this in vitro dermal absorption study in human epidermis, a dermal absorption value of 2.00% was found for the concentrate and 19.13% for the highest dilution.

Executive summary:

In an OECD TG 428 study in compliance with GLP the absorption and distribution of test substance from an EC formulation was measured in vitro through human epidermis. The doses were applied as the formulation concentrate (nominally 125 g / L) and as two aqueous spray dilutions (1/83 v/v and 1/1250 v/v) of the formulation in water. The doses were applied to the epidermal membranes at a rate of 10 μL/cm2 and the weight recorded. The cells were left unoccluded for an exposure period of 24 hours. The absorption process was monitored using [14C]-labelled test substance, which was added to a blank formulation and water as appropriate, prior to application. The distribution of test substance within the test system was investigated and a 24 hour absorption profile was determined. The samples were analysed by LSC.

The absorption rate of the concentrate between 0 - 8 hours was 0.037 µg/cm2/h, after which it increased to 0.066 µg/cm2/h between 8 - 16 hours. Fastest absorption occurred between 16 - 24 hours, when test substance was absorbed at a rate of 0.085 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.061 µg/cm2/h. For the 1/83 v/v aqueous spray dilution, The absorption rate between 0 - 6 hours the absorption rate was 0.008 µg/cm2/h, after which it increased to 0.016 µg/cm2/h between 6 - 12 hours. Fastest absorption occurred between 12- 24 hours, when test substance was absorbed at a rate of 0.018 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.015 µg/cm2/h. For the 1/1250 v/v aqueous spray dilution, test substance absorption was essentially linear throughout the entire 24 hour exposure period. Between 0-24 hours the mean absorption rate was 0.003 µg/cm2/h.

Absorption was calculated according the Efsa guidance on dermal absorption (2017). Following the test conditions, dermal absorption is considered as the sum of the concentration in the Stratum corneum, remaining epidermis and receptor fluid. Cells with recovery rates below 90% or 110% were excluded from the calculation. To include an uncertainty factor on variance a multiplication factor based on the number of cells was multiplied with the standard deviation of the average dermal absorption observed. This results in the following dermal absorption values: 2.00, 6.10 and 19.13% for the concentrate, 1/83 dilution and 1/1250 dilution, respectively.

Based on this in vitro dermal absorption study in human epidermis, a dermal absorption value of 2.00% was found for the concentrate and 19.13% for the highest dilution.