Trisodium 1-(1-naphthylazo)-2-hydroxynaphthalene-4',6,8-trisulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 10 to 13, 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:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Red 018

IUPAC Name:

Acid Red 018

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

PREPARATION
- Procedure used: on day of receipt, tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of pre-incubation before treatment was 60 min.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room T, 35 min at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: after 60 ± 1 min from start of exposure, tissues were rinsed with PBS and transferred to a fresh medium; after 24 ± 20 min of post-incubation period, the medium was replaced by a fresh-one
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Libra S22
- Wavelength: 570 nm
- Filter: no external filter
- Filter bandwidth: 2-3 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment, composed of 3 replicate tissues

PREDICTION MODEL / DECISION CRITERIA
- In case the test chemical is found to be non-corrosive (e.g. based on guideline 430, 431 or 435) and tissue viability after exposure and post-treatment incubation is less than (<=) 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS category 2.
- The test 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

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg atop previously moistened tissue with 25 µl PBS

POSITIVE CONTROL
- Concentration: 5 % SDS in aqueous solution

Duration of treatment / exposure:

60 minutes: 25 minutes at room temperature and 35 minutes at culture conditions

Duration of post-treatment incubation (if applicable):

24 h and 20 minutes, then medium was replaced by a fresh one and tissues were incubated for 19 h and 7 minutes.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

90.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (OD570 is 1.682)
- Acceptance criteria met for positive control: yes (% of negative control tissues in 2.9 %)
- Acceptance criteria met for variability between replicate measurements: yes (SD of 3 identically treated replicates is < 18 % in all cases)

Any other information on results incl. tables

Average viability of treated tissues was 91.4 % of negative control viability. Negligible OD570 was observed in colorant control tissues extract though correction could be done.

True tissue viability = 91.4 % - 1.1 % = 90.3 %.

OD570 values obtained at MTT test, averages, standard deviations (%) and relative viabilities

	treatment	OD570			avg	SD	average viability (% NC)
		1	2	3
NC	PBS	1.752	1.728	1.567	1.682	0.082	100
	% viab. NC	104.1	102.7	93.1	100	4.882
C1	PBS	1.529	1.601	1.483	1.538	0.049	91.4
	%	90.9	95.2	88.2	91.4	2.887
C1 CC	medium, no MTT	0.019	0.018	-	0.018	0.001	1.1
	%	1.129	1.011	-	1.1	0.059
PC	5 % SDS	0.065	0.069	0.066	0.067	0.002	4.0
	%	3.9	4.1	3.9	4.0	0.101

C1 test substance

CC colorant control

NC negative control

PC positive control

SD standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: non classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

Average tissue viability upon treatment with test substance is 90.3 %.
Effect of test substance in EpiDermTM model was negative.

Executive summary:

Method

In vitro skin irritation study according to OECD guideline 439. One single experiment was run, using 3 replicates for test substance, positive and negative control. After pre-incubation of tissues, 25 mg test substance were spread on the tissue surface. Exposure duration was 60 minutes. After substance removal, tissues were post-incubated for ca. 42 hours. Afterwards, 3 hours incubation with MTT and 3 hours extraction followed. Optical density (OD570) of isopropyl alcohol was measured on a spectrophotometer.

Controls for colour interference and direct MTT reduction were also carried out.

Results

Slight colour interfernce and no direct MTT reduction were found.

Average cell viability was 90.3 %, i.e. above 50 %. Therefore, effect of the substance in EpiDermTM model was negative.