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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:
other: OECD Guideline for the Testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22, 2010)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide
EC Number:
229-419-9
EC Name:
2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide
Cas Number:
6528-34-3
Molecular formula:
C18H18N4O6
IUPAC Name:
2-[(4-methoxy-2-nitrophenyl)diazenyl]-N-(2-methoxyphenyl)-3-oxobutanamide
Test material form:
solid: bulk

Test system

Amount / concentration applied:
10 mg of the undiluted test item were applied to three EPISKIN (Skinethic) tissues, test item 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:
10 µL of the undiluted test item, the positive control (5% sodium lauryl sulfate) and the negative control (deionised water) were applied to three EPISKIN (Skinethic) tissues. 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 69 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
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
all
Value:
>= 92.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After treatment with the test item the relative absorbance values did not decrease relevantly (92.3%; 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 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

1.095

1.046

1.148

1.096

99.9

95.4

104.7

4.7

100.0

Positive Control

15 min

0.441

0.383

0.340

0.388

40.2

34.9

31.0

4.6

35.4

Test Item

15 min

0.935

1.024

1.077

1.012

85.2

93.4

98.2

6.5

92.3

*       Mean of two replicate wells after blank correction

**      relative absorbance per tissue [rounded values]
***
    relative absorbance per treatment group [rounded values]

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

The relative absorbance value of the test item, corresponding to the cell viability, did not decrease (threshold for irritancy:≤50%), consequently the test item was non irritant to skin.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
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 skin irritation potential of the test item by means of the Human Skin Model Test.


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


Approximately 10 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 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 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 as compared to the negative control to 35.4% 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 7% (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 (92.3%; threshold for irritancy:50%). Therefore, the test item is not considered to possess an irritant potential.


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

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