Tetrasodium 2-[[4-[[4-[[1-hydroxy-6-(phenylamino)-3-sulphonato-2-naphthyl]azo]-1-naphthyl]azo]-6-sulphonato-1-naphthyl]azo]benzene-1,4-disulphonate

Endpoint summary

Description of key information

Skin Irritation

In Vitro test Skin irritation was choosen as a key study. The test was performed according totheOECDTest GuidelineNo.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.

GLP study.

Klimish score 1.

Eye Irritation

In vivo test Eye Irritation was choosen as a key study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.03.-02.06.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 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. 25802 kit C)- Date of initiation of testing: 30.6.2016TEMPERATURE 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 gNEGATIVE CONTROLPBS MatTek 101816ZSA, exp. 18/10/2017 - Amount(s) applied: 25 µLPOSITIVE CONTROL5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 031617MGKA, exp. 16/03/2018

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

101.1

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT reduction - functional check in tubes The test was not performed due to blue colour of the test substance. The next step was performed directly.

Table No. 1: OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities 

	Treatment	OD570			Avg	SD	Average viability
Code		1	2	3			(% NC)
NC	PBS	1.961	1.946	1.941	1.949	0.008	100.0
	% of avg NC	100.6	99.8	99.6	100.0	0.4
C4	595/16	2.093	2.047	1.775	1.972	0.140	101.1
	% of avg NC	107.4	105.0	91.1	101.1	7.2
C4 CC	595/16	0.010	0.004	-	0.007	0.003	0.4
	% of avg NC	0.5	0.2	-	0.4	0.2
PC	5% SDS	0.059	0.053	0.056	0.056	0.002	2.9
	% of avg NC	3.0	2.7	2.9	2.6	0.1

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 78 was 101.1 % of negative control average value i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model. According to the classification criteria given in chapter 4.5 of this report, the test substance, Direct Blue 78, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.

Executive summary:

The test substance, Direct Blue 78, was assayed for in vitro skin irritation in the human epidermal model EpiDerm^TM. The test was performed according to the OECD Test Guideline No.439:In Vitro Skin 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).

Colour interference was performed simultaneously. The test substance did not interfere with the endpoint.

Direct MTT reduction in the test tube was not performed because of blue colour of solutions of the test substance. Direct MTT test in frozen tissues excluded direct MTT reduction caused by the test substance. 

After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissuesurface. 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.

Under the above-described experimental designaverage viability of treated tissues was101.1%,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 given in chapter 4.5. of this report,the test substance, Direct Blue 78, is considered to have no category in regard to skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.-03.1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards and acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

The study report contains the limited information on the test conditions because the study has been performed in 1991 . However available information is sufficient to conclude on the classification of the substance. Further the test was performed on the vertebrates and use of results from old experimental studies is one of the options to provide information requested by REACH. New experimental studies with vertebrates must only be conducted if there is no adequate existing information.

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Controls:

not specified

Irritation parameter:

other: cornea damage

Basis:

mean

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

other: mucosa demage

Basis:

mean

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is nonirritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on available data the test substance, Direct Blue 78, is not classified as a skin irritant.

Based on available data the test substance, Direct Blue 78, is not classified as a eye irritant.