Sodium 4-amino-5-hydroxy-3-(4-nitrophenylazo)-6-(phenylazo)naphthalene-2,7-disulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 4 to 7, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted on a Similar Substance. The reliability of the original report is 1. The complete justification for the read across approach is attached at section 13.

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)

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 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

223-098-9

EC Name:

Disodium 5-acetylamino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonate

IUPAC Name:

Acid Red 001

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Vehicle:

other: PBS (phosphate buffered saline)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, USA)
- Tissue batch number(s): Lot No. 23361- Tissue surface: 0.63 cm²The EpiDerm™ tissues are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were rinsed with PBS, blotted to remove the test substances, and then transferred to fresh medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3h
- Spectrophotometer: Libra S22
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
Three tissues are used per the test substance, three for the the positive (PC) and three for the negative (NC) controls.

DIRECT MTT REDUCTION - functional check in tubes
- the test substance is added to 1 mL MTT medium (red) and incubated in the incubator at culture conditions for 1 hour.
- At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.

COLOUR INTERFERENCE - coloured substances
25 mg of the test substance are added to 2 ml isopropanol/water and placed on an orbital plate shaker for 2 to 3 hours at room temperature. After centrifugation, two 200 μl aliquots of supernatant and pure isopropanol (blank) are transferred to a 96-well plate and the absorbance is measured with a plate reader at OD570.
If, after subtraction of the OD for isopropanol, the OD of the test article solution is
> 0.08 the material has to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls has to be performed.
Each coloured test article is applied to two additional tissues (the colorant controls (CC)) and is treated in the same way. Instead in MTT medium, these tissues are kept in assai medium. The following steps are identical as in the MTT test.
The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSCliving).
True viability = %Viability of treated tissue – %NSCliving

EVALUATION OF THE RESULTS AND CLASSIFICATION
For further classification the relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %. The cut-off values for the prediction of irritation are given below; these values are stated in OECD Test Guideline No. 439 (1), par. 36:
a. In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2.
b. The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of thr test substance

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

approximately 42 hours

Number of replicates:

3
reconstructed human epidermal model EpiD

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 85.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Colour interference
25 mg of the test substance were added to 2 mL of water and another 25 mg were added to 2 mL of isopropyl alcohol in the test tube. The test substance was partially soluble in water (red solution) and a little soluble in isopropyl alcohol (pink solution).
Both tubes were shaken for 120 minutes at room temperature and then centrifuged. Two 200 μl aliquots of every supernatant and pure isopropanol (blank) were transferred to a 96-well plate and OD was measured at 570 nm.
Water extract was non-measurable. OD570>3.
Pure OD570 of isopropyl-alcohol extract was 0.029 what is <0.08.
It means that, at good rinse of the dissolved test substance from tissues, colorant control has not to be performed.

Direct MTT reduction-functional check in tubes
25 mg of the test substance was added to 1.0 mL of MTT medium. Solution was incubated for 1 hour (37±1°C, 5±1 % CO2, humidified). The test substance changed colour
of MTT medium to red,). The test substance is not directly-reducing.

MTT test
Duration of pre-incubations before treatment was 60 minutes and 19 hours and 58 minutes.
25 mg of the test substance was placed directly atop the tissue moistened with 25 µL of PBS. The material was then spread on the tissue surface. Length of exposition was 60 minutes of which 25 minutes were tissues kept at room temperature and the remaining 35 minutes at culture conditions. After that the test substance was removed, tissues were rinsed and post-incubated. 1st post-incubation took 23 hours 48 minutes and 2nd took 18 hours, 21 minutes.
The other handling was performed as described in paragraph 4.3.3.
The dissolved test substance was rinsed from tissues - no colour was eluted to medium at post-incubations. Small amount of the test substance remained in tissues and coloured them red. Tissues remained coloured red even after extraction with isopropyl alcohol (see Figure 2).
The third tissue was perforated together with its pad at removal of the test substance from tissue surface what markedly influenced on tissue character at the experiment and decrease of OD570.

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Conclusions:

Not irritant

Executive summary:

Method

The test substance, was assayed for the in vitro skin irritation in human epidermal model EpiDerm^TM.The test was performed according to the OECD Test Guideline No. 439.

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

 

Results

Under the above-described experimental design average viability of treated tissues was 85.5 %, i.e. viability was > 50 %.

Conclusion

The test substance is considered to have no category in regard to skin irritation.

Not irritant