Reaction mass of 5,5'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[3-methylsalicylic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and potassium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxy-7-sulfonaphthalene-2-sulfonate - 2,2',2''-nitrilotriethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs®, 10 mm ø) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 23747 (Certificate of Analysis see apendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):50 µl bulk volume (about 70 mg)

Details on study design:

To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.Due to the intense color of the test substance it was not possible to evaluate whether or not the test substance is able to reduce MTT directly, therefore freeze-killed control tissues (KC) were treated with the test article and the negative control in the same way.
The color of a test substance may interfere with the color density produced by metabolic capacity of the tissue and would falsify the test results when residues of the test substance remain on the tissues after washing and are extracted by the isopropanol. Due to the color of the test substance a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as follows: the test substance was applied to a KC tissue, incubated and removed by washing. Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically. Based on the result of the pretest it was judged that application of color control tissues is not necessary.
Several test substances were tested in parallel within the present test (test no. 81) using the same control tissues (NC and PC).
Two tissues were treated with each, the test substance, the PC and the NC. In addition two killed tissues were used for each, the test substance and the NC, in order to detect direct MTT reduction. There are two separate protocols for liquids and solids, differing in exposure time and post-incubation period. Due to the physical state of the test substance the protocol for solids was applied. On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours. After the pre-incubation, the tissues were pre-treated with 20 µL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes. The test substance could not be applied with a sharp spoon. Therefore a metal pin was covered with a bulk volume of ca. 50 µL of the undiluted test material and was applied with direct contact to the tissue. Control tissues were concurrently applied with 50 µL of sterile de-ionized water (NC, NC KC) or with 50 µL of methyl acetate (PC) or test substance (KC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed. To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) in order to remove residual test substance.
After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period). After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
Negative control (NC): De-ionized water, sterile
Positive control (PC): Neat methyl acetate (CAS No.:79-20-9)

Results and discussion

In vitro

Results

Remarks on result:

other: The mean viability o fthe test-substance treated tissues was 80.8%.

Any other information on results incl. tables

Test substance identification			tissue 1	tissue 2	mean	Inter-tissuee variability[%]
NC	viable tissues	mean OD570	1.506	1.469	1.487
		viability [% of NC]	101.2	98.8	100.0	2.5
	KC tissues	mean OD570	0.033	0.025	0.029
		viability [% of NC]	2.2	1.7	2.0	0.5
16/0255-1	viable tissues	mean OD570	1.282	1.32	1.207
		viability [% of NC]	86.2	76.1	81.2	10.1
	KC tissues	mean OD570	0.004	0.008	0.006
		viability [% of NC]	0.3	0.5	0.4	0.2
	Final mean viability of tissues after KC correction[% of NC]:				80.8
PC	viable tissues	mean OD570	0.152	0.209	0.181
		viability [% of NC]	10.2	14.1	12.2	3.8

 

The tissues were black discolored and compound residues remained on the tissues after the washing procedure.

Due to the intense color of the test substance, the ability of the test substance to reduce MTT directly could not be determined in a pretest. Therefore KC tissues were applied in parallel.

The results of the KC tissues indicate an increased MTT reduction (mean viability 0.4% of NC). Thus for the test substance the final mean viability is given after KC correction.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that Direct Black 18L NA active dye does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.