Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Direct Black 51- Molecular formula (if other than submission substance): C27H17N5Na2O7S- Molecular weight (if other than submission substance): 601.5- Substance type: organic- Physical state: solid- Analytical purity: cca 80 % w/w- Impurities (identity and concentrations): cca 20 % NaCl (CAS: 7647-14-5)- Lot/batch No.: 9013- Storage condition of test material: 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:

non-transformed keratinocytes

Details on animal used as source of test system:

TISSUESThe reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Ashland, USA); Lot No. 21675, kit A) has been obtained from MatTek Bratislava

Vehicle:

physiological saline

Details on test system:

PROCEDUREOn the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After pre-incubations (1 and 18±3hours), tissues were topically exposed to the test chemicals for 1 hour. Three tissues were used per the test substance (C1), for the positive (PC) and negative (NC) controls. Tissues were then thoroughly rinsed, blotted to remove the test substance, and transferred to fresh medium. After 24±2 hours incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18±2 hours. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 mL/tissue of isopropyl alcohol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M/48/3 (VUOS-CETA, 2011).OD570 MEASURINGOD570 (optical density at 570 nm) was measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank.NEGATIVE CONTROLThe absolute optical density of the negative control (NC) tissues (treated with sterile phosphate buffered saline) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8.POSITIVE CONTROLA 5% solution sodium dodecyl sulphate in water is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95±1 % confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied: 25 mg

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Test system

Vehicle:

unchanged (no vehicle)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

94.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

see Table No. 1

Any other information on results incl. tables

Table No. 1: Viability values

	Treatment	OD(570)			Mean	SD	Relative viability (% NC)
		1	2	3
NC	PBS	2.076	2.058	1.934	2.023	0.063	100.0
	viability (% NC)	102.6	101.7	95.6	100.0	3.12
C1	270/15	1.767	2.065	1.897	1.910	0.122	94.4
	viability (% NC)	87.4	102.1	93.8	94.4	6.031
PC	5% SDS	0.058	0.049	0.062	0.056	0.005	2.8
	viability (% NC)	2.9	2.4	3.1	2.8	0.269

NC - negative control

PC - positive control

C1 - test substance

SD - Standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Average viability of tissues treated by the test substance was 94.4 % 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 method, the test substance, Direct Black 51, is considered to have no category in regard to skin irritation.

Executive summary:

Test substance, Direct Black 51, was assayed for the in vitro skin irritation in human epidermal model EpiDerm™. The test was performed according to the EU Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDerm™ Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

Average viability of tissues treated by the test substance was 94.4 %, i.e.viability was > 50 %.

The effect of the test substance was negative in EpiDerm™ model (the tissue was not damaged).

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