Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From the 21th of April to the 2th of May, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted according to an internationally accepted test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

deviations are not considered to have affected the integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

EC Number:

274-403-7

EC Name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Cas Number:

70210-06-9

Molecular formula:

C31H25N5O6S2.2Na

IUPAC Name:

disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: epidermis keratinocytes

Source strain:

other: human recontructed epidermis

Test animals

Strain:

other:

Details on test animals or test system and environmental conditions:

EPISKIN™
Commercial Name EPISKIN™ - 0.38 cm2
Supplier SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batch 16-EKIN-017 (alive tissues) and 15-EKIN-050 (killed tissues)
Arrived at RTC on 26 April 2016 and 15 Dec 2015
Quality controls: histology scoring, magnitude of viability and barrier function (IC50 determination).
Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.

PREPARATION OF THE TEST ITEM
Examination at arrival
Temperature indicator: pale grey (suitable for use)
pH indicator: orange (suitable for use)
Preparation and pre-treatment incubation period
The test system was shipped on Monday and received on Tuesday. According to the supplier procedure, tissues were prepared as follows:
– Alive tissues: at arrival, plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium.
Culture plates were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.
– Killed tissues: a sufficient number of epidermis units were placed in a 12-well plate in which each well had previously been filled with 2 mL/well sterile water for injection.
Tissues were incubated for approximately 48 hours, then transferred into a new plate and stored at -20°C.
The day of the experiment, tissues were thawed at room temperature with 2 mL of maintenance medium.

Test system

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

other: Maintenance medium, assay medium

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20 mg/epidermis unit (14.6 mg of active constituent/epidermis unit)

Duration of treatment / exposure:

An exposure time of 15±0.5 minutes was allowed in a ventilated cabinet at room temperature.
Due to the difficulties encoutered in removing the test item, for negative control tissues and for one of the epidermis treated with the positive control, the exposure period was prolonged (approximately 16-19 minutes).

Observation period:

A 42 ± 1 °C hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

Details on study design:

Preliminary test
Direct MTT reduction test (Step 1): Non-specific reduction of MTT was evaluated as follows: two mL of MTT ready-to-use solution (0.3 mg/mL) was incubated with 20 mg of test item at 37°C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance
of the solution at the end of the incubation time was carried out.
Colouring potential test (Step 2): Chemicals’ colouring potential was assessed for potential interaction with the test system. 20 mg of the test item was added to 180 µL of distilled water (Baxter; batch no. 15D2902) in a transparent tube and the resulting solution/suspension mixed by using a vortex for
15 minutes. Colouring of the solution/suspension at the end of the incubation time was evaluated by spectral analysis at 595 nm.

Main Assay
Alive tissues were treated with the test item, positive and negative controls.Due to the test item features, the epidermis surfaces were moistened wi h 10 µL of sterile water after application of the test item, with the exception of one epidermis (B1), which was moistened before the test item application.

Washing
At the end of the exposure, each negative and positive control tissue was rinsed with approximately 25mL of sterile D-PBS, while the test item was washed four times with 25mL of sterile D-PBS, filling and empting the tissue insert, in order to completely remove the test item from the epidermis.
The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2mL/well of maintenance medium.

Each tissue insert was incubated with 2 mL/well of MTT ready-to-use solution. Plates were incubated for approximately 3 hours at 37°C, 5% CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were preserved for approximately 3 days at 4°C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD)
values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range, an MTT formazan calibration curve was performed.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

83.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean cell viability of the test item treated tissues, after the appropriate background subtraction, was 83.5%.
Based on the results obtained, the test item is classified as not irritant to the skin.

Any other information on results incl. tables

PRELIMINARY TEST

In a first step, the test item was assayed for the ability of reducing MTT per se. A dark red coloured solution, without precipitation, was observed in the MTT suspension at the end of the incubation period, indicating that the test item could interact with MTT.

In a second step, the test item was assayed for the ability of colouring water per se. A dark red coloured solution was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed.

The value obtained for the Optical Density (OD) was 2.898, indicating that the test item has a potential interfering

ability.

Based on the results obtained, additional controls were added in the Main Assay for the evaluation of Non Specific Colouring potential (NSCliving) and Non Specific MTT reduction (NSMTT).

Since the test item was able both to stain tissue and reduceMTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.

MAIN TEST

The mean Optical Density of Blank Controls was 0.040, lower than the maximum acceptable value (0.1).

The test item induced an overall interference, calculated as [(NSMTT + NSCliving) - NSCkilled], of 0.255%. Based on this result, only the OD-blank background subtraction was performed and the mean cell viability was 83.5%, when compared to the negative control.

Acceptable intra-replicate variability was obtained (SD of % viability = 10.4 lower than 18). It should be noted that, due to difficulty in removing the test item, the NSCkilled value was 13.69% , higher than the NSMTT (7.86%); thus the overall interference may be underestimated.

However, the mean cell viability of test item treated epidermis corrected for the NSCliving and for NSMTT values was higher than 50% (data not presented, but retained in the study file).

Therefore the subtraction of the NSCkilled value to the overall interference did not affect the evaluation of the test item irritation properties.

The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

Positive control results indicated an appropriate cell death with an acceptable relative cell viability (6.6% of the negative control value). Variability between replicates gave also the expected value (SD of %viability = 0.7). Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.

Applicant's summary and conclusion

Interpretation of results:

other: not classified

Remarks:

Classification criteria according to the CLP Regulation 1272/2008 and its amendments

Conclusions:

Mean cell viability of the test item treated tissues, after the appropriate background subtraction, was 83.5%.
Based on the results obtained, the test item is considered not irritant to the skin.

Executive summary:

Method

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439, in GLP.

A preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. In a second step, the test item was assayed for the ability of colouring water per se.

Based on the results, additional controls were added in the Main Assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit (14.6 mg of active constituent/epidermis unit), with positive and negative controls.

Observations

The test item did not induce cell death in any replicate, the mean cell viability after appropriate subtraction was 83.5% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 10.4 (lower than 18, as stated in the Study Protocol).

Conclusions

The substance is considered not irritant to the skin.