N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 439), performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22, 2010)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

EC Number:

235-426-8

EC Name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Cas Number:

12225-08-0

Molecular formula:

C27H24N6O7S

IUPAC Name:

4-{[2,5-dimethoxy-4-(methylsulfamoyl)phenyl]diazenyl}-3-hydroxy-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Test system

Amount / concentration applied:

Each approximately 10 mg of the neat test item were applied to three EPISKIN (Skinethic) tissues. Additionally, the tissues were wetted with 15 µL of deionised water.

Duration of treatment / exposure:

The skin equivalents were exposed to the test item for 15 minutes. After completion of the treatment the test item was rinsed off and the skin equivalents were incubated for further 42 hours.

Details on study design:

Each approximately 10 mg of the neat test item and 10 µL of the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate, SLS) were applied to each three Episkin (Skinethic) tissues. Additionally, the test item treated tissues were wetted with 15 µl of deionised water. The test item as well as the controls were rinsed off after 15 minutes treatment. After further 42 hours incubation the tissues were treated with the MTT solution for 3 hours following approximately 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined phototmetrically at a wavelength of 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: relative absorbance value

Value:

>= 0.867 - <= 1.034

Remarks on result:

other:

Remarks:

Basis: other: three Episkin (Skinethic) tissues. Time point: After treatment with the test item. Remarks: 91.6% (threshold for irritancy: = 50%).

Other effects / acceptance of results:

After treatment with the test item the mean relative absorbance value was reduced to 91.6% (threshold for irritancy: = 50%). Therefore, the test item is not considered to possess an irritant potential.

Any other information on results incl. tables

Results after treatment with test item and controls

 

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	1.106	0.909	1.011	1.009	109.6 90.1 100.2	9.8	100.0
Positive Control	15 min	0.242	0.297	0.193	0.244	24.0 29.5 19.1	5.2	24.2
Test Item	15 min	1.034	0.867	0.872	0.924	102.5 86.0 86.5	9.4	91.6

*       Mean of two replicate wells after blank correction

**      relative absorbance per tissue [rounded values]: (100 x absorbance of tissue) / mean absorbance of negative control
***    relative absorbance per treatment group [rounded values]: (100 x absorbance of test item) / mean absorbance of negative control

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

 

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 91.6% (threshold for irritancy:=50%). Consequently the test item was non irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test according to OECD TG 439.

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes.

Each about 10 mg of the test item were applied to the tissues, wetted with 15 µL of deionised water, and spread to match the tissue size.

10 µL of either the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) were applied to each tissue.

The test item and the positive and negative controls were washed off the skin tissuesafter 15 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following approximately 72 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD greater or equal 0.6 till less or equal 1.5 for the15 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance compared to the negative control to 24.2%, thus ensuring the validity of the test system.

The standard deviations between the % variabilities of the test item, the positive and negative controls were below 10% (threshold of the "OECD TG 439: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value was reduced to 91.6% (threshold for irritancy:=50%). Therefore, the test item is not considered to possess an irritant potential.