Reaction products of diazotized Dodecyl Aniline coupled with 8-acetylamine-1-hydroxynaphthalen-3,6-disulphonic Acid, sodium salts

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 29.Mar.2017 to14.Apr.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

EC Number:

946-819-5

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human derived epidermis keratinocytes

Cell source:

other: reconstructed human epidermis (RhE)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: model, EPISKIN™
- Tissue batch: 17-EKIN-015 (alive tissues); 17-EKIN-012 (killed tissues)
- Shipping date: 10 April 2017
- Delivery date: 11 April 2017
- Date of initiation of testing:
- Source: SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)

PREPARATION OF THE TEST SYSTEM
-Examination at arrival
Temperature indicator: pale grey (suitable for use)
pH indicator: orange (suitable for use)

-Preparation and pre-treatment incubation period
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.

-Media
Maintenance Medium SkinEthic; batch: 17-MAIN3-014
Assay Medium SkinEthic; batches: 17 ESSC 011 and 17-ESSC-014

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure, each tissue was rinsed with approximately 25 ml of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 ml /well of maintenance medium.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
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.

NUMBER OF REPLICATE TISSUES: 3

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Negative control (Live tissue) D-PBS 20 µL
Positive control (Live tissue) 5% (w/v) SDS 20 µL
Test item (Live tissue) 20 ± 2 mg
Test item without MTT (NSCliving) (Live tissue) 20 ± 2 mg
Test item (Killed tissue) 20 ± 2 mg
Negative control (Killed tissue) D-PBS 20 µL
Test item without MTT (NSCkilled) (Killed tissue) 20 ± 2 mg

Duration of treatment / exposure:

An exposure time of 15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

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

Number of replicates:

Negative control (Live tissue) D-PBS: 3
Positive control (Live tissue): 3
Test item (Live tissue): 3
Test item without MTT (NSCliving) (Live tissue): 2
Test item (Killed tissue): 2
Negative control (Killed tissue) D-PBS: 2
Test item without MTT (NSCkilled) (Killed tissue): 2

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: mean cell viability %

Value:

105

Negative controls validity:

valid

Positive controls validity:

valid

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 bordeaux suspension, with a dark bordeaux precipitate, was observed at the end of the incubation period when the test item was added to the MTT solution, indicating that the test item could direct interact with MTT.

In a second step, the test item was assayed for the ability of colouring water per se. A dark bordeaux suspension 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 2.874, indicating that the test item has a potential interfering ability.

Based on these results, additional controls (NSC and NSMTT) were added in the Main Assay.

Main Assay

The mean Optical Density of Blank Controls was 0.039, lower than the maximum acceptable value (0.1). The negative control gave the expected baseline value (mean Optical Density value ≥0.6 and ≤1.5) 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 (2% of the negative control value). Variability between replicates gave also the expected value (SD of % viability = 0.1). 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.

The colouring interference (NSC) was 1%, as well as the non specific MTT reduction (NSMTT), thus only the blank subtraction was performed.

The test item did not induce cell death in any replicate, the mean cell viability after the appropriate subtractions was 105% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.2.

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

The test item did not induce cell death in any replicate, the mean cell viability after the appropriate subtractions was 105% when compared to the negative control.

Executive summary:

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 were based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. A preliminary test was carried out to evaluate the compatibility of the test item with the test system. The value obtained for the Optical Density (OD) indicated that the test item has a potential interfering ability. Based on these 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 ± 2 mg/epidermis unit, each measuring 0.38 cm² (treatment level: 53 mg/cm²). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µl/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSC killed) was performed.

The colouring interference (NSC) was 1 %, as well as the non specific MTT reduction (NSMTT); thus only the black subtraction was performed. The test item did not induce cell death in any replicate, the mean cell viability after the appropriate subtractions was 105%, when compared to the negative control. All the validity criteria were met. Based on the results obtained, the test item is not classified as irritant to the skin (UN GHS No Category).