Disodium 7-[[4,6-bis[(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 08th, 2015 to January 22nd, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The test was performed according to internationally accepted guidelines and in accordance with internationally valid GLP principles.

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)

Version / remarks:

Adopted: 28th July, 2015

Deviations:

yes

Remarks:

not impacting the test results

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Direct Red 253

IUPAC Name:

Direct Red 253

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:

The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed human epidermis (RhE) model.
- Viable tissues: the reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia)
Lots Nos. 23306 and 23308
- Frozen tissues: the reconstructed human epidermal model EpiDerm™ killed by freezing
Lot No.: 16888 FRZN EA

DIRECT MTT REDUCTION - functional check in tubes
50 mg of the test substance was added to 2.0 ml of MTT medium. Solution was incubated for 1 hour (37 ± 1 °C, 5 ± 1 % CO2, humidified).

DIRECT MTT REDUCTION - test in frozen tissues
The test substance (25 mg) was applied to three freeze-killed tissues for 60 min exposition. In addition, two freeze-killed tissues were treated with PBS for 60 min. After 60 min of incubation (37 ± 1 °C, 5 ± 1 % CO2, moistened), the test substance was rinsed off and tissues were incubated with MTT solution in the same manner as viable tissues in MTT test. Two hours extraction in isopropylalcohol with shaking and OD measuring at 570 nm followed consequently.

MTT VIABILITY ASSAY
The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 25 µl of PBS. The material is spread on the tissue surface.
A single testing, composed of three replicate tissues, was run.
On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubations durable for approximately 1 and 18 hours, tissues are topically exposed to the test chemicals for 1 hour (25 minutes at room temperature and the remaining 35 minutes at 37 °C, 5 % CO2). Three tissues are used per the test substance, for the positive (PC) and negative (NC) controls. Tissues are then thoroughly rinsed with PBS, blotted to remove the test substances, and transferred to fresh medium.
After 24 ± 2 hours post-incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18 ± 2 hours. Afterwards, the MTT assay is 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 is extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm.

OD570 MEASURING
OD570 is measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank. Allowed band width is 2-3 nm. No external filter is used.

VIABILITY CALIBRATION
Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test substance is calculated – this value is used for the comparison with limit.

ASSAY ACCEPTANCE CRITERIA
When any of the acceptance criteria is not met the experiment has to be repeated.

Negative Control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) 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
OD570 historical negative control range is 1.470-2.342.

Positive Control
A 5 % SDS (in H2O) solution 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 %.
OD570 historical positive control range is 0-0.203.

Standard Deviation (SD)
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18 %.

EVALUATION OF RESULTS
In vitro alternatives that have been validated and accepted may also be used to help in classification decisions making (Regulation (EC) 1272/2008, 3.2. Skin corrosion/ irritation, 3.2.2. Classification criteria for substances).
The cut-off values for the prediction of irritation are given below:
- 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 %.
A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5 %, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Amount/concentration applied:

25 mg of substance/surface ratio 39.7 mg/cm2

Duration of treatment / exposure:

1 hour (25 minutes at room temperature and the remaining 35 minutes at 37 °C)

Duration of post-treatment incubation (if applicable):

24 ± 2 hours

Number of replicates:

Three replicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

> 50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT TEST - VIABILITY ASSAY
In the first experiment tissues could be damaged at washing: wiping and multiple washing together with longer staying out of media probably decreased the cell viability in one of the three replicates.
Criterium #3 of assay acceptance criteria for SD among tissues was not fulfilled in the first experiment. The first experiment was then repeated.

In the second experiment longer time gaps were let between treatment of tissues to obtain more time for rinsing and tissues were not wiped, only rinsed. In the second experiment the first tissue of negative control was lost by technical mistake during processing. Therefore, no results from this tissue have been acquired.

Direct MTT reduction-functional check in tubes: the test substance changed colour to red.
Direct MTT reduction - test in frozen tissues: average OD570 value of treated tissues after 60 min treatment (0.055) was lower than that in negative control (PBS-0.058), so there is no interference with evaluation and correction of results of MTT test is not necessary.

EVALUATION OF RESULTS AND CLASSIFICATION
Under the above-described experimental design, average viability of tissues treated by the test substance was 88.1 % 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, the test substance considered as non-irritant to skin.

ACCEPTANCE CRITERIA FULFILMENT
Criterium #3 for SD of among tissues was fulfilled neither in the first nor in the second experiment.
The first experiment was repeated and its results were not taken into account for the final evaluation.
In the second experiment range of SD in % of test item was also higher than 18 % (18.8 %). As also the lowest viability was above 50 % cut-off value, this experiment was accepted and evaluated.
The other criteria were fulfilled.

Any other information on results incl. tables

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

First experiment

	Treatment	OD570			Mean	SD	Average viability (NC %)
		1	2	3
NC	PBS	1.984	1.910	1.879	1.924	0.044	100.0
	%	103.10	99.26	97.64	100.00	2.289
C1	432/15	0.259	1.094	1.553	0.969	0.536	50.3
	%	13.46	56.85	80.70	50.34	27.836
PC	5 % SDS	0.056	0.077	0.070	0.068	0.009	3.5
	%	2.91	4.00	3.64	3.52	0.454

Second experiment

	Treatment	OD570			Mean	SD	Average viability (NC %)
		1	2	3
NC	PBS	0.179*	1.855	1.832	1.844	0.012	100.0
	%	9.71*	100.62	99.38	100.00	0.624
C3	432/15	1.201	1.622	2.049	1.624	0.346	88.1
	%	65.15	87.98	111.15	88.09	18.779
PC	5 % SDS	0.075	0.065	0.069	0.070	0.004	3.8
	%	4.07	3.53	3.74	3.78	0.223

NC: negative control; PC: positive control; C1, C3 TS: test substance; *tissue lost during processing excluded from evaluation; mean: arithmetic mean; SD: standard deviation calculated from individual % tissue viabilities: viability (%): viability of single tissues compared with negative control; NT: not tested; NE: not evaluated

Direct MTT reduction in frozen tissues

Treatment	Tissues			Average	SD	NC %
	1	2	3
PBS 60 min	0.055	.0.60	NT	0.058	0.003	100.0
%	95.7	104.3	NT	100.0	4.3	-
432/15	0.054	0.056	0.054	0.055	0.001	95.1
%	93.9	97.4	93.9	95.1	1.6	-

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to CLP Regulation (EC 1272/2008)

Conclusions:

Non skin irritating.

Executive summary:

Test substance 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.

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance 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.

As the test item is coloured red, test with frozen tissues was performed to detect if the test item remaining in tissues interfered with measuring of OD₅₇₀. Interference was not evidenced.

First time, average viability was 50.3 %, what is near of cut-off value and high SD among tissues was observed. So the experiment was repeated and average viability of treated tissues was 88.1 %, i.e.viability was > 50 %.

The effect of the test substance was negative in EpiDermTM model (tissues were not damaged).

Conclusion

The test substance is considered to be non skin irritating.