Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date - 02 September 2015; Experimental completion date - 07 September 2015; Study completion date - 19 December 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Deviations:

yes

Remarks:

See below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See below

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

EC Number:

257-854-4

EC Name:

Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

Cas Number:

52333-30-9

Molecular formula:

C47H38N6O14S4.2Na

IUPAC Name:

disodium 5,5'-[propane-2,2-diylbis(4,1-phenyleneoxysulfonyl-2,1-phenylenediazene-2,1-diyl)]bis(6-amino-4-hydroxynaphthalene-2-sulfonate)

Test material form:

other: solid

Specific details on test material used for the study:

Identification: FAT 21021/E TE
Batch: 72
Purity: ≥75 %
Physical state/ Appearance: Red solid
Expiry date:16 February 2020
Storage Conditions: room temperature in the dark.

In vitro test system

Test system:

human skin model

Remarks:

EPISKIN™ Reconstructed Human Epidermis Model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN Reconstructed Human Epidermis Model Kit
- Tissue batch number: 15 EKIN 035
- Date of initiation of testing:
02 September 2015.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
One

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.2 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: UV Vis
- Wavelength: 562nm

NUMBER OF REPLICATE TISSUES: Triplicate

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 10 mg (26.3 mg/cm²) of the test item was applied

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours incubation at 37 °C.

Number of replicates:

Three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

105.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

See "Any other information on results incl. tables"

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT. 

 

Assessment of Color Interference with the MTT endpoint

Colored test items may interfere with the MTT endpoint. Therefore, an additional functional procedure using viable color correction tissues was performed to measure any potential interference. However, the results obtained showed that negligible interference due to the color of the test item occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 9.9 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.1 %. The positive control acceptance criterion was therefore satisfied. The mean OD₅₆₂ for the negative control treated tissues was 0.718 and the standard deviation value of the viability was 1.0 %. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.6 %. The test item acceptance criterion was therefore satisfied.

 

Mean OD₅₆₂Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562of tissues	Mean OD562of triplicate tissues	±SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.726	0.718	0.007	101.1	100*	1.0
	0.715			99.6
	0.712			99.2
Positive Control Item	0.064	0.071	0.008	8.9	9.9	1.1
	0.080			11.1
	0.069			9.6
Test Item	0.766	0.755	0.012	106.7	105.1	1.6
	0.755			105.2
	0.743			103.5

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100 %

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results

The relative mean viability of the test item treated tissues was 105.1% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

 

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU DSD and CLP Not classified for Irritation.

UN GHS Not classified for Irritation (Category 3 cannot be determined).