Methyl 4-[[(2,5-dichlorophenyl)amino]carbonyl]-2-[[2-hydroxy-3-[[(2-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]benzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 MAR 2012 to 02 APR 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 439) and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Original Guideline adopted July 22, 2010)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide

EC Number:

229-245-3

EC Name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)azo]-N-phenylnaphthalene-2-carboxamide

Cas Number:

6448-95-9

Molecular formula:

C24H18N4O4

IUPAC Name:

3-hydroxy-4-[(2-methyl-5-nitrophenyl)diazenyl]-N-phenyl-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): In Vitro Skin Irritation Test: Human Skin Model Test: Novoperm Red RT-531-D-MX
Analytical purity: 89.2 % (w/w)

Test system

Amount / concentration applied:

Approximately 11 mg of the neat test item were applied to three EPISKIN (Skinethic) tissues. Additionally, the tissues were wetted with 20 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:

Approximately 11 mg of the neat test item and 10 µL of the negative control (deionised water) or the positive control (5% sodium lauryl sulfate) were applied to each three EPISKin (Skinethic) tssues. Additionally, the test item treated tissues were wetted with 20 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 70 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at a wavelength of 570 nm.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Relative Absorbance [%]

Value:

107.2

Results 2

Irritation / corrosion parameter:

other: Relative Absorbance [%]

Value:

93.1

Results 3

Irritation / corrosion parameter:

other: Relative Absorbance [%]

Value:

96.4

Any other information on results incl. tables

Results after treatment with test substance 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	1.107	1.040	1.124	1.090	101.5 95.4 103.1	4.0	100.0
Positive Control	15 min	0.321	0.304	0.336	0.320	29.4 27.9 30.8	1.4	29.4
Test Item	15 min	1.169	1.015	1.051	1.078	107.2 93.1 96.4	7.4	98.9

*       Mean of two replicate wells after blank correction

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

After treatment with the test item the relative absorbance values did not decrease relevantly (98.9%; threshold for irritancy: =50%). Therefore, the test item is not considered to possess an irritant potential.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

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 tets substance 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.

Approximately 11 mg of the neat test item were applied to each tissue, spread to match the tissue size. Additionally, the test item tissues were wetted with 20 µL of deionised water.

The test item and the positive and negative controls were washed off the skin tissues after 15 minutes treatment. After further incubation for about 42.5 hours the tissues were treated with the MTT solution for 3 hours following 69.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

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

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD in the range from 0.6 to 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 as compared to the negative control to 29.4% 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 8% (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%) thus ensuring the validity of the study.

After treatment with the test item the relative absorbance values did not decrease relevantly (98.9%; threshold for irritancy:=50%). Therefore, the test item is not considered to possess an irritant potential.