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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-09 to 2015-03-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
Paris, April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Environmental Health and Safety Publications, Series on Testing and Assessment no. 28. Guidance document for the conduct of skin absorption studies
Version / remarks:
Paris, March 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guidance Notes on Dermal Absorption, Series on Testing and Assessment No. 156, ENV/JM/MONO(2011)36.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.45 (Skin Absorption: In Vitro Method)
Version / remarks:
Brussels, May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EFSA Panel on Plant Protection Products and their Residues (PPR); Guidance on Dermal Absorption. EFSA Journal 2012;10(4):2665.
Deviations:
no
Principles of method if other than guideline:
The study was designed to examine the in vitro percutaneous absorption of Imazalil, formulated as Imazalil sulphate though human and rat skin membranes. The test substance was tested at two target concentrations: 75g.Imazalil/L (concentrate) and 0.25 g/L (field dilution). The concentrate represents the maximal concentration possible when hendling the undiluted formulation, while the low concentration reflects the concentration recommended for use in the field. The objective of the study was to elucidate the extent of percutaneous absorption of the compound-related radioactivity. The contact time was 8 hours, i.e. a normal working day, and the post exposure time was 16 hours. In addition to the amount of [14C]Imazalil in the receptor fluis, the residues remaining in/on the skin membranes and in the stratum corneum (16h post exposure) were determined. The study was performed in flow-through diffusion cells.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-[2-(allyloxy)ethyl-2-(2,4-dichlorophenyl)-1H-imidazolium hydrogen sulphate
EC Number:
261-351-5
EC Name:
1-[2-(allyloxy)ethyl-2-(2,4-dichlorophenyl)-1H-imidazolium hydrogen sulphate
Cas Number:
58594-72-2
Molecular formula:
C14H14Cl2N2O.H2O4S
IUPAC Name:
1-[2-(2,4-DICHLOROPHENYL)-2-(PROP-2-EN-1-YLOXY)ETHYL]-1H-IMIDAZOLE SULFATE (1:1)
Test material form:
solid: particulate/powder
Details on test material:
- Product names: FUNGAFLOR® 75C / FUNGAFLOR® 75SP / FUNGAZIL® 750SP / FUNGAFLOR® 75PS / FUNGAZIL® 75SP / FUNGAFLOR® 750SP
- Reference number: JNJ-2634372-ABI / R027180 (product code)
- Physical state: solid
- Appearance: beige solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: UB027180EXA132
- Expiration date of the lot/batch: 2015-09-25
- Purity test date: 2015-02-20

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: 98.1 %
- Specific activity: 56.4 mCi.mmol-1, 6.98 MBq.mg-1)
- Locations of the label: 14C
- Expiration date of radiochemical substance: 2020-03-05 (recertification date)
- Storage conditions: <-18°C

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature (15-25 ºC)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified
- Other: The homogeneity of [14C]Imazalil in the dose preparations was checked; the coefficients of variation (CV) of the dose preparations were 0.4% (A/C) and 1.3% (B/D), and therefore considered sufficient.


FORM AS APPLIED IN THE TEST (if different from that of starting material)
Concentration a.i. : 76.3 g.L-1
Density : 1.0329 g.mL-1

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : BA027180 (Fecundal® 7.5 S / Imazalil sulphate 100 SL)
Radiolabelling:
yes

Test animals

Species:
other: human and rat skin membranes

Administration / exposure

Type of coverage:
other: skin membranes placed in diffusion cells
Vehicle:
unchanged (no vehicle)
Duration of exposure:
8h
Doses:
- Actual doses:
Group A (human): 76.64 g.L-1 (Mean dose a.i. applied (μg.cm-2) 792 ± 2.4)
Group B (human): 0.25 g.L-1 (Mean dose a.i. applied (μg.cm-2) 2.51 ± 0.03)
Group C (rat): 76.64 g.L-1 (Mean dose a.i. applied (μg.cm-2) 798 ± 0.7)
Group D (rat): 0.25 g.L-1 (Mean dose a.i. applied (μg.cm-2) 2.53 ± 0.01)

- Rationale for dose selection: not specified

- Dose preparations:
Test group Amount of [14C]Imazalil Amount of concentrate formulation Amount of demi water Total concentration measured (g/L) Radioactive concentration measured (MBq.mL-1)
A/C 235 μL (~2.6 MBq, ~0.37 mg) 1.0321 g - 76.64 2.37
B/D 171 μL (~1.75 MBq, ~0.25 mg) - 1.0001 g 0.25 1.71

In the formulations, Imazalil is present as the sulphate salt. Therefore, in addition to the amount of [14C]Imazalil, in both formulations an equimolar amount of sulphate was added. To this purpose, a stock solution of H2SO4 in demineralised water was prepared by mixing 0.1052 g H2SO4 with 19.9043 g of demineralised water.
For dose formulation A/C, first 0.0249 g of H2SO4 stock solution was added to a brown glass vial after which the target amount of [14C]Imazalil, dissolved in ethanol, was added. The solvent was evaporated under N2 gas until complete dryness and 1.0321 g of concentrate formulation was added.
For test concentration B/D, first 0.0169 g of H2SO4 stock solution was added to a brown glass vial after which 157 μL of [14C]Imazalil, dissolved in ethanol, was added. The solvent was evaporated under N2 gas until complete dryness and 1.0001 g of demineralised water was added. Following overnight stirring, the radioactive concentration was found to be 9.4% under target. Therefore, an additional 14 μL of [14C]Imazalil in ethanol, was added to a clean brown glass vial. The solvent was evaporated under N2 gas until complete dryness and the complete amount of dose B/D was added.
The dose preparations were kept at ambient temperature until application while stirring.
No. of animals per group:
Groups A/B: Two skin membranes from four donors in each test group
Groups C/D: Two skin membranes from three donor rats in each test group
Control animals:
yes
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin:
Human skin membranes were prepared from frozen skin samples, present at TNO Triskelion. Human skin, derived from the breast and abdomen, was obtained from four female donors directly after surgery.
Rat skin was obtained from three male rat (Wistar WU, Charles River, Germany) of ca. 12 weeks old. After the sacrifice of the rat, the dorsal and flank skin of the animal was clipped free of fur by means of electric clippers. After collection on 17 December 2014 (code: R14/24 - 26), the skin was stored at <−18 °C until use.
- Ethical approval if human skin: not specified
- Preparative technique: Upon thawing, the skin was cut using a Dermatome. The thickness of all skin preparations was measured with a digimatic micrometer.
- Thickness of skin (in mm): 0.2-0.4 mm
- Membrane integrity check: yes
- Storage conditions: stored at < −18 °C until use
- Justification of species, anatomical site and preparative technique: according to guideline

PRINCIPLES OF ASSAY
- Diffusion cell: 9 mm flow-through automated diffusion cells
- Receptor fluid: saline (0.9% sodium chloride (w/v)) containing 0.01% sodium azide (w/v), supplemented with 6% polyoxy-ethylene 20-oleyl glycol (PEG, w/v), pH 7.4.
- Solubility of test substance in receptor fluid: The solubility of Imazalil in water was reported to be ca. 180 μg.mL-1. The maximum absorption into the receptor fluid was 138 μg (i.e. 215 μg.cm-2) in ca. 40 mL over 24 h, i.e. 3.4 μg.mL-1. Therefore, the solubility of the test substance in the receptor fluid was considered sufficient, especially since 6 % polyoxy-ethylene 20-oleyl glycol (w/v) was added to the receptor fluid to further improve the solubility.
- Static system: not described
- Flow-through system: flow rate of ca. 1.8 mL.h-1
- Test temperature: recorded at 15-minute intervals, 32 ± 1 ºC
- Humidity: ambient humidity
- Occlusion: no
- Reference substance(s): [3H]H2O

Results and discussion

Percutaneous absorptionopen allclose all
Time point:
24 h
Dose:
792 ± 2.4 (μg.cm-2)
Parameter:
percentage
Absorption:
2.8 %
Remarks on result:
other: Potentially absorbed dose
Remarks:
Group A Human
Time point:
24 h
Dose:
798 ± 0.7 μg.cm-2
Parameter:
percentage
Absorption:
21.7 %
Remarks on result:
other: Potentially absorbed dose
Remarks:
Group C Rat
Time point:
24 h
Dose:
2.51 ± 0.03 μg.cm-2
Parameter:
percentage
Absorption:
27.3 %
Remarks on result:
other: Potentially absorbed dose
Remarks:
Group B Human
Time point:
24 h
Dose:
2.53 ± 0.01 μg.cm-2
Parameter:
percentage
Absorption:
41.4 %
Remarks on result:
other: Potentially absorbed dose
Remarks:
Group C Rat

Any other information on results incl. tables

Integrity of skin membranes

Prior to the determination of the percutaneous absorption of Imazalil, the permeation coefficient (Kp) for tritiated water was determined in human and rat skin membranes.

Skin membranes with a Kp value below the cut-off value of 2.5×10-3 cm.h-1 (human) or 3.5×10-3 cm.h-1 (rat) were selected for the study.

Human skin

The mean absorption of Imazalil from the concentrate formulation into the receptor fluid over the 24 h study duration was 4.07 μg.cm-2, representing 0.51 % of the applied dose. The mean maximal flux for the absorption of Imazalil through human skin was 0.24 μg.cm-2.h-1 and the lag time was 0.2 h. Some replicates showed an increase in the cumulative absorption curve beyond the 8-hour washing step. When the maximal flux values for these replicates were calculated beyond eight hours, the mean maximum flux value was slightly higher, i.e. 0.28 ± 0.17 μg.cm-2.h-1.

The mean absorption of Imazalil from the field dilution into the receptor fluid was 0.46 μg.cm-2, representing 18.2 % of the dose applied.

The mean maximal flux through human skin was 0.041 μg.cm-2.h-1 and the lag time was 1.6 h.

The mean total absorption, defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash and the skin membranes (excluding tape strips)

was 1.7 ± 1.2 % (concentrate formulation) and 24.3 ± 11.2 % (field dilution) of the applied dose.

The mean potentially absorbed dose, which is defined as the compound-related radioactivity present in the receptor fluid, the receptor compartment wash, the skin membranes and the

stratum corneum (except for the first 2 tape strips) was 2.8 ± 1.6 % (concentrate formulation) and 27.3 ± 11.9 % (field dilution) of the applied dose.

The mean recovery of Imazalil in human skin was 99.5 +- 4.2 % and 97.1 +- 4.5 % for the concentrate formulation and field dilution, respectively.

For both the concentrate formulation and the field dilution, less than 75 % of the absorption of Imazalil in the receptor fluid over 24 hours occurred within half of the study duration (i.e. 12 hours).

For risk assessment, in agreement with the EFSA Guidance Document on Dermal Absorption (2012), it is considered appropriate to include the tape strips (except the first 2 tape

strips) in the calculations of the total absorption values (i.e. the potentially absorbed dose).

Rat skin

The mean absorption of Imazalil from the concentrate formulation into the receptor fluid over the 24 h study duration was 134 μg.cm-2, representing 16.8 % of the applied dose. The mean maximal flux for the absorption of Imazalil through rat skin was 42.2 μg.cm-2.h-1 and the lag time was 0.1 h.

The mean absorption of Imazalil from the field dilution into the receptor fluid was 0.86 μg.cm-2, representing 34.1 % of the dose applied.

The mean maximal flux through rat skin was 0.089 μg.cm-2.h-1 and the lag time was 0.8 h.

The mean total absorption through rat skin was 20.1 ± 5.6 % (concentrate formulation) and 38.9 ± 4.9 % (field dilution) of the applied dose.

The mean potentially absorbed dose was 21.7 ± 5.4 % (concentrate) and 41.4 ± 3.9 % (field dilution) of the applied dose.

The mean recovery of Imazalil in rat skin was 99.3 +- 2.8 % and 99.7 +- 2.5 % for the concentrate formulation and field dilution, respectively.

Applicant's summary and conclusion

Conclusions:
Based on the maximal flux, for the concentrate formulation human skin was 178 times less permeable for Imazalil compared to rat skin (42.2 / 0.24), while for the field dilution, human skin was 2.2 times less permeable for Imazalil compared to rat skin (0.089 / 0.041).
For the concentrate formulation, based on mean absorbed dose, human skin was 11.6 times less permeable for Imazalil compared to rat skin (20.1 / 1.7).
For the field dilution, human skin was 1.6 times less permeable for Imazalil compared to rat skin (38.9 / 24.3).
Based on the potentially absorbed dose, for the concentrate formulation human skin was 7.9 times less permeable for Imazalil compared to rat skin (21.7 / 2.8), while for the field dilution, human skin was 1.5 times less permeable for Imazalil compared to rat skin (41.4 / 27.3).

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