4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 - 22 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

No information provided on technical proficiency; no positive control data after 3 min exposure

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

No information provided on technical proficiency; no positive control data after 3 min exposure

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 Bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

EC Number:

227-030-9

EC Name:

4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

Cas Number:

5610-94-6

Molecular formula:

C₄₃H₂₂N₆O₁₃S₃

IUPAC Name:

4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container closed and dry in a cool, well-ventilated place, protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human derived non-transformed epidermal keratinocytes

Source strain:

other: not applicable

Justification for test system used:

The reconstructed human epidermis model test method is an accepted in vitro method to replace in vivo animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Remarks:

; the epidermis surface was wettened with 20 ± 2 µL of deionised water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Skin Ethic™ RHE-model RHE/S/17, manufactured by EPISKIN/Skin Ethic Laboratories, Lyon, France
- Tissue batch number: 17-RHE-021
- Date of initiation of testing: 21 Feb 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume of washing steps: gently rinsed with approx. 20 mL DPBS solution


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h (± 15 min)
- Spectrophotometer: 96-well plate microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 570 = 1.3 (OD 570 historic negative control: 1.881 (3 min exposure) and 2.001 (1 h exposure)
- Barrier function: 4.6 h
- Morphology: 5.5 cell layers, well-differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue viability after 3 min exposure is < 50%, or the mean tissue viability is ≥ 50% after 3 min exposure and < 15% after 1 h exposure.
(If the mean tissue viability after 3 min exposure is < 18% the substance is considered as corrosive Sub-categories 1A and if it is ≥ 18% the substance is considered as combination of optional Sub-categories 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability is ≥ 50% after 3 min exposure and ≥ 15% after 1 h exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 ± 3 mg

NEGATIVE CONTROL
- Amount(s) applied: 40 ± 3 µL
- Concentration: neat/ undiluted

POSITIVE CONTROL
- Amount(s) applied: 40 ± 3 µL
- Concentration: 8N

Duration of treatment / exposure:

3 min and 1 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of test item (100%), 3 min exposure

Value:

100.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean value of test item (100%),1 h exposure

Value:

108.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information given

ACCEPTANCE OF RESULTS:
The mean OD 570 value of the negative control tissues should be between 0.8 and 3. The negative controls should be higher or equal to a historical established boundary (here: two standard deviations below the current historical mean = 1.635 (1.881 - 2 x 0.123) and 1.429 (2.001 - 2 x 0.286) for 3 min and 1 h exposure respectively.

The positive controls should be lower or equal to a historical established boundary (here: three standard deviations above the current historical mean = 0.99% (0.58 + 3 x 0.14).

The range between identically treated tissues viability should be < 30% with the exception of cases with ODs ≤ 0.3 and for viabilities out the range 20 - 100%.

- Acceptance criteria met for negative control: Yes. The mean OD value of the two negative control tissues was in the range of 0.8 - 3 at each exposure time (2.238 and 2.020 at 3 min and 1 h exposure, respectively).
- Acceptance criteria met for positive control: Yes. The positive control treatment resulted in a viability of < 0,99% (0.6% at 1 h exposure).
- Acceptance criteria met for variability between replicate measurements: Yes. In the range 20 - 100 % viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates did not exceed 30% in any case (negative control: 9% after 3 min exposure and 0.6% after 1 h exposure, treatment group: 10.5% and 2.1% after 3 min and 1 h exposure, respectively). For the positive control the threshold of 30% was exceeded (40%), but as the OD 570 was < 0.3 it is not considered to be a deviation.

Any other information on results incl. tables

Table 1: MTT assay after 3 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD570	2.142	2.334	-	-	2.138	2.361
OD570 (mean values of replicates)	2.238		-		2.25
Viability (%)	100		-		100.5

Table 2: MTT assay after 1 h exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD570	2.027	2.013	0.01	0.014	2.174	2.22
OD570 (mean values of replicates)	2.02		0.012		2.197
Viability (%)	100		0.60		108.8

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model according to OECD Guideline 431. 

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 +/- 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide) were applied to the tissues. Before application of 20 +/- 3 mg of the solid test item, 20 +/- 2 µL of deionised water was spread to the epidermis surface to improve the contact between test item and the epidermis. 

After treatment with the positive control, the mean viability value was 0.6% and, thus, lower than the historically established threshold of 0.99%. After treatment with the negative control (deionised water) the mean ODs were 2.238 (3 minute exposure) and 2.020 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.635 and 1.429, respectively. Thus, the acceptance criteria were met. 

Following treatment with the test item, the tissue viability was >= 50% after 3 minutes exposure (mean viability: 100.5%) and >= 15% after 1 hour exposure (mean viability: 108.8%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.