Climbazole

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-12 to 2010-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to EU-Method B.46 " In vitro Skin Irritation: Reconstructed human epidermis model test" (23. July 2009)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: According to Human Skin Model Test EU-Method B.46 adopted 23. July 2009

Deviations:

no

Principles of method if other than guideline:

The in vitro skin irritation test is used to determine the skin irritancy of chemicals by measuring the cell viability in a reconstructed human epidermis (RhE) model after applying the test substance. The RhE model consists of human-derived epidermal keratinocytes, which have been cultured to form a multilayered model of the human epidermis. Irritant chemicals are identified by their ability to decrease cell viability by quantitatively measuring the enzymatic conversion of the vital dye MTT (3-[4,5-dimethyl thiazole 2-yl] 2,5-diphenyl-tetrazoliumbromide) to a blue formazan salt. Skin irritation potential of the test substance is assessed by reducing the formazan production to less than 50% of the negative control.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Climbazole

EC Number:

253-775-4

EC Name:

Climbazole

Cas Number:

38083-17-9

Molecular formula:

C15H17ClN2O2

IUPAC Name:

1-(4-chlorophenoxy)-1-(1H-imidazol-1-yl)-3,3-dimethylbutan-2-one

Test animals

Species:

other: not applicable (this is an in vitro test)

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

other: not applicable (this is an in vitro test)

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable (this is an in vitro test)

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Amount of test item: mean: 22.7 mg

Duration of treatment / exposure:

60 min

Observation period:

not applicable (cell viability was determined after the end of treatment period)

Number of animals:

not applicable (this is an in vitro test)

Details on study design:

The human skin model test was performed with commercially available Epi-200-SIT-Kit. The EpiDermTM tissues were produced from MatTek Corporation in Ashland, USA. The day of delivery was the 01. July 2010 batch: 13657

Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) without CaCl2 and MgCl2
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer
Positive control: Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in deionised H2O, concentration 50 g/L
One plate (three tissues) was used as positive control; each tissue was treated with 30 µL SDS-solution
Test item: Crinipan AD, ground before use; 22.7 mg for each tissue
One plate (three tissues) was used for treatment with the test item.The tissues were wetted with 25 µL DPBS before applying the test item

Experimental performance:
After dosing the tissues with negative control, positive control and test item, they were incubated at 37 °C for 35 min. After 60 min a rinsing step and two incubation periods at 37°C over a total period of 42 h followed.
After incubation, a MTT assay was performed and formazan production was measured by a spectral photometer at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60min

Value:

112.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: percentage values of formazan production in comparison to the negative control

Other effects / acceptance of results:

not applicable
The mean relative absorbance value was increased to 112.7% after the treatment with Crinipan AD. This value is well above the threshold for irritation (50%). Therefore, the test item is considered not irritant. The mean reduction of the relative absorbance of the positive control was 9.0%.

Any other information on results incl. tables

Comparison of formazan production:

For the test item and the positive control the following percentage values of formazan production were calculated in comparison to the negative control:

% Formazan Production

Designation	Crinipan AD	Positive control
% Formazan production (Tissue 1)	93.9%	9.4%
% Formazan production (Tissue 2)	130.5%	7.5%
% Formazan production (Tissue 3)	114.4%	10.3%
% Formazan production Mean	112.7%	9.0%

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance did not show any skin irritation properties in the EpiDerm reconstituted human skin model.

Executive summary:

This in-vitro study human skin model test (EU method B.46) was performed in order to evaluate the potential of crinipan AD to evoke skin irritation in a human-skin-model. The test item crinipan was spread on the surface of three sections of an EpiDerm^TM tissue and incubated for 60 min. The negative control consisting of Dulbecco’s phosphate buffered saline was applied to another three tissue sections. As positive control a SDS-solution (50 g/L) was used for three other tissue sections. After rinsing and two incubation steps of altogether 42 h an enzyme assay to determine cell viability was performed. The tissues were incubated with MTT-reagent for 3 h. The tissues were washed with PBS, dried and transferred to a 24 -well-plate. After incubating with isopropanol at room temperature over night, the developed formazan concentration was measured in a spectral photometer at 570 nm.

The mean percentage values of formazan production were calculated in comparison to the negative control. The mean relative absorbance value was increased to 112.7% after the treatment with crinipan. This value is well above the threshold for irritation (50%). The positive control showed a mean reduction of formazan production of 9.0%.

In conclusion, it can be stated that the test item crinipan is considered as non skin irritant under the experimental conditions used in this study.