4-(2-phenylpropan-2-yl)phenyl 3-diazo-4-oxo-3,4-dihydronaphthalene-1-sulfonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 for 3 min exposure, 1 h exposure at room temperature (instead of 37°C)

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 for 3 min exposure, 1 h exposure at room temperature (instead of 37°C)

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-(2-phenylpropan-2-yl)phenyl 4-oxo-3-(λ⁵-diazynylidene)-3,4-dihydronaphthalene-1-sulfonate

Cas Number:

23121-00-8

Molecular formula:

C25H20N2O4S

IUPAC Name:

4-(2-phenylpropan-2-yl)phenyl 4-oxo-3-(λ⁵-diazynylidene)-3,4-dihydronaphthalene-1-sulfonate

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

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace 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:

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 (3 min or 1 h)

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
Isopropanol was used to extract the formazan salt from the tissues. The plates were put on a shaker (VWR Microplate Shaker, VWR GmbH, Darmstadt, Germany) with gentle agitation of about 120 rpm at room temperature for 2 h (± 5 min).

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 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 per exposure time and per group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

(of negative control)

Run / experiment:

mean value of test item, 3 min exposure

Value:

98.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

(of negative control)

Run / experiment:

mean value of test item, 1 h exposure

Value:

116.3

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

ACCEPTANCE OF RESULTS:
- 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 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). Regarding the positive control the threshold of 30% was exceeded (40%), but as the OD 570 was < 0.3 it is not considered as 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	-	-	2.091	2.301
OD570 (mean values of replicates)	2.238		-		2.196
Viability (%)	100		-		98.1

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	0.010	0.014	2.331	2.368
OD570 (mean values of replicates)	2.020		0.012		2.350
Viability (%)	100		0.60		116.3

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.