Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.04-29.07.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

EC Number:

277-616-3

EC Name:

Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Cas Number:

73816-74-7

Molecular formula:

C28H24ClN7O10S3.3Na

IUPAC Name:

trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reactive Red 45:1
- Physical state: red solid powder
- Analytical purity: 85% (w/w)
- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 15% (w/w)
- Lot/batch No.: 7010
- Expiration date of the lot/batch: unlisted
- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: model EpiDermTM
- Tissue: The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23329)
- Date of initiation of testing: 19.04.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 g

NEGATIVE CONTROL
PBS (phosphate buffered saline) prepared 25/02/2016, exp. 25/08/2016 and prepared 23/03/2016 exp. 23/09/2016
- Amount(s) applied: 25 µL


POSITIVE CONTROL
5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 012616TMB, exp. 26/01/2017

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

54.3

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test item Reactive Red 45:1 was 54.3 % of negative control average value. The measured value was then corrected, due to red colour of the test substance.
Corrected viability of treated tissues was 53.4 %, i.e. viability was > 50 %.

Executive summary:

The test item, Reactive Red 45:1, 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: In VitroSkin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

After pre-incubation of tissues, 25 mg of the test item was placed directly a top to the previously moistened tissue and it was spread on the entire tissuesurface. Length of exposition was 60 minutes. Three tissues were used for the test item and every control.

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.

At direct reduction assay in a test tube MTT medium was coloured red, so direct MTT reduction was excluded.

In the first experiment too high differenceamong viability of tissues was observed in range close to cut off value. The experiment was then repeated. In the repeated experiment all test conditions were fulfilled and average viability of treated tissues was54.3% (53. 4 % after correction),i.e. viability was >50 %.

The effect of the test item was negative in EpiDerm^TMmodel (tissues were not damaged).

According to the classification criteria given in chapter 4.5., the test item, Reactive Red 45:1, is considered to have no category in regard to skin irritation.