Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

disodium 4-amino-3-[2-(4-{4-[2-(2,4-diaminophenyl)diazen-1-yl]benzenesulfonamido}phenyl)diazen-1-yl]-5-hydroxy-6-(2-phenyldiazen-1-yl)naphthalene-2,7-disulfonate

EC Number:

605-104-5

Cas Number:

157577-99-6

Molecular formula:

C34H26N10Na2O9S3

IUPAC Name:

disodium 4-amino-3-[2-(4-{4-[2-(2,4-diaminophenyl)diazen-1-yl]benzenesulfonamido}phenyl)diazen-1-yl]-5-hydroxy-6-(2-phenyldiazen-1-yl)naphthalene-2,7-disulfonate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

Test System
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, Slovakia) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

Quality control
The developer/supplier provided demonstration that each batch of the RhE model used meets acceptable production release criteria – tissue viability, barrier function, sterility and barrier function after post refrigerated storage.
Demonstration of proficiency in performing the test method before routine use was performed by testing of the proficiency substances.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The negative control is DPBS (Dulbecco’s phosphate buffered saline). 30 μL of PBS will be placed onto 2 tissues for 60±1 minutes exposure.
The positive control is 5% SDS (sodium dodecyl sulphate) water solution. 30 μL of the positive control are applied onto the 2 tissues for 60±1minutes exposure.
Controls are tested concurrently with the application form in every run performed by a single technician.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 96.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD570 of the NC tissue was 1.950 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8. This value is within historical negative control limits 1.403-2.304.
The mean viability of the PC tissues expressed as % of the negative control tissues was 1.7 % which meets the acceptance criterion of ≤ 20 %. OD570=0.033 is within historical positive control limits 0-0.176.
The SD calculated from individual % tissue viabilities of the identically treated replicates was 0.1 % for the positive control, 11.1 % for negative control and 10.54 % for the test item what is < 18 % in all cases.
All study acceptance criteria were fulfilled

Any other information on results incl. tables

Direct MTT Reduction - Functional Check in Tubes

After incubation of 25 mg of the test item with 1 mL MTT medium (red) for about 1 hour at culture conditions, MTT medium did not change its colour towards blue/purple. Resulting suspension was coloured dark green.

The test item did not reduce MTT directly.

Colour Interference

Part of the test item remained in tissues after washing, after post-incubations and even after extraction with isopropyl alcohol. The tissues were coloured green.

Average OD570 of colorant control tissues was 0.029, what is <5% of negative control viability. In this case the correction of results not have to be performed.

The colour of the test item did not interfere with the endpoint

MTT test

No changes in appearance of tissues treated with the test item were observed during the experiment. Tissues were dry, smooth and coloured dark green.

Average OD570 of extracts from colorant control tissues was 1.5%, what it was less than 5% of the negative control viability. In such case correction of results is not necessary

Applicant's summary and conclusion

Interpretation of results:

other: non skin irritant

Conclusions:

Under the above-described experimental design of the MTT test, the average viability of tissues treated with the test item Acid Black 234 was 96.2±10.5% of the negative control average value i.e. viability was > 50 %. The positive control, SDS, gave the appropriate response.
The test item colour does not influence study results and the test item does not have direct reductive properties, so any correction of MTT test results was not performed.
The effect of the test item was negative in the EpiDermTM model.
According to the classification criteria, the test item is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant to skin.

Executive summary:

The test item, Acid Black 234, was assayed for in vitro skin irritation in the human epidermal model EpiDerm^TM. The main methodical documents were OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed HumanEpidermisTest Method(2019) andProtocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In the main MTT experiment, after pre-incubation of tissues, 25 mg of the test item was placed directly on the moistened tissue and spread over the entire surface for 60 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Two tissues were used for the test item and for positive and negative controls. Besides, two tissues were treated in the same way due to confirmation/refutation of colour interference of the test item (colorant control).

After removal of the test item, tissues were post-incubated in culture medium for approximately 42 hours. After three hours incubation with MTT (colorant control tissues were incubated in medium), samples were extracted with isopropyl alcohol for 2 hours and 25 minutes. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Part of the test item remained in tissues after washing and after post-incubations. So tissues treated with the test item were coloured green. Some green colour was extracted with isopropyl alcohol into extracts, but it was less than 5% of the negative control viability, so that correction was not necessary.

In the other complementary experiment, direct MTT reductionin test tubes, direct reduction was not found. Therefore, the results of the MTT test did not require correction.

Under the above-described experimental design, theaverage viability of test item-treated tissues was 96.2±10.5%.

The positive control, SDS, gave the appropriate response.

 

The test item was negative in the EpiDermTMmodel (i.e. not irritating).

According to the classification criteria,the test item, Acid Black 234, is considered to have no category in regard to skin irritation.