Tetrasodium 8-[[4-[(4-amino-3-sulphonatophenyl)azo]-6-sulphonatonaphthyl]azo]-5-[[6-(benzoylamino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-2-sulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4.10.2016 - 20.1.2017

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: 28th July, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 761/2009, 23rd July 2009

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol for: INVITRO EpiDermTM SKIN IRRITATION TEST For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm

Version / remarks:

Model EPI-200-SIT, Rev. 26/3/2012,1-37

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 8-[[4-[(4-amino-3-sulphonatophenyl)azo]-6-sulphonatonaphthyl]azo]-5-[[6-(benzoylamino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-2-sulphonate

EC Number:

274-427-8

EC Name:

Tetrasodium 8-[[4-[(4-amino-3-sulphonatophenyl)azo]-6-sulphonatonaphthyl]azo]-5-[[6-(benzoylamino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-2-sulphonate

Cas Number:

70210-31-0

Molecular formula:

C43H30N8O14S4.4Na

IUPAC Name:

tetrasodium 8-[[4-[(4-amino-3-sulphonatophenyl)azo]-6-sulphonatonaphthyl]azo]-5-[[6-(benzoylamino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-2-sulphonate

impurity 1

Reference substance name:

unknown impurities

IUPAC Name:

unknown impurities

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: 7031/2007Stability/Expiration: Feb 2018Storage: The test substance should be stored in dry room in dark in supplied container at the room temperature.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: tissue for research puposes from accredited institutions

Source strain:

other: Keratinocyte strain 00267

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUEViable tissues: the reconstructed human epidermal model EpiDerm (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23361 kit DFrozen tissues: the reconstructed human epidermal model EpiDermTM killed by freezing; Lot No.: 23354 kit DTEMPERATURE USED FOR TEST SYSTEMculture conditions 37±1°C, 5±1 % CO2, moistened tissueREMOVAL OF TEST MATERIAL AND CONTROLS- Volume and number of washing steps: thoroughly rinsed with PBS, blotted to remove the test substanceMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg·mL-1- Incubation time: 180±5 mins- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank. FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATABased on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.NUMBER OF REPLICATE TISSUES: 3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- killed tissues- Procedure used to prepare the killed tissues (if applicable): by freezing- N. of replicates : 2NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:Direct MTT reduction - functional check in tubes - not performed due to the test substance intrinsic blue colour. 1. Colour interference => not use concurrent colorant control in the MTT test2. MTT test (frozen tissues) => the correction of result was not necessary3. MTT test (viable tissues)PREDICTION MODEL / DECISION CRITERIAOECD Test Guideline No. 439 (1), par. 36:- 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. - 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) was placed directly on tissue moistened with 25 µL of PBS and spread on the tissue surface. NC: PBS (phosphate buffered saline), prepared in laboratory 30/08/2016 (live tissues), exp. 02/03/2017MatTek 092316MHE (frozen tissues); exp. 23.9.2017 PC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 020616TMB in water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

101.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All study acceptance criteria were fulfilled. The mean OD570 of the NC tissue was 2.373 ±0.115 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.The mean viability of the PC tissues expressed as % of the negative control tissues is 3.3% which meets the acceptance criterion of ≤ 20 %. The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance was < 18 %.

Any other information on results incl. tables

MTT test (frozen tissues)

Treatment	tissue 1	tissue 2	mean	SD	%NC
water (NC)	0.049	0.041	0.045	0.004
avg OD/%	4.1	3.4	3.7	0.33	100.0
79/16 (C1)	0.049	0.055	0.052	0.003
avg OD/%	4.1	4.6	4.3	0.25	115.6

Average TS OD₅₇₀value has been a bit higher than average OD₅₇₀value of the NC. Nevertheless, SDs (97.5% confidence interval) of both measuring have overlapped so correction of result is not necessary.

MTT test (viable tissues)

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

Treatment	OD570			Mean	SD	%NC
PBS (NC)	2.450	2.458	2.211	2.373	0.115
viability (%NC)	103.2	103.6	93.2	100.0	4.8	100.0
79/16 (C1)	2.168	2.619	2.431	2.406	0.185
viability (%NC)	91.4	110.4	102.4	101.4	7.8	101.4
5% SDS (PC)	0.063	0.087	0.082	0.077	0.010
viability (%NC)	2.7	3.7	3.5	3.3	0.4	3.3

PBS - phosphate buffered saline

SDS - sodium dodecyl sulphate

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 substance Direct Blue 85 was 101.4 % of negative control average value, i.e. viability was > 50 %. The effect of the test item was negative in EpiDermTM model (tissues were not damaged).According to the classification criteria the test substance, Direct Blue 85, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.

Executive summary:

The test item, Direct Blue 85, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method test (2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (2012).

In preliminary experiment no colour interference with the endpoint was found.

Direct MTT reduction of test substance was not solved by a test in test tube due to its blue colour. Therefore test in frozen tissues was performed directly.

After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately 42 hours. 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.

No signs of direct reduction were found in the test in frozen tissues, so result obtained in MTT test was not corrected.

Under the above-described experimental design average viability of treated tissues was 101.4%, i.e. viability was >50 %.

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

According to the classification criteria, the test substance, Direct Blue 85, is considered to have no category in regard to skin irritation.