1,1'-[(6-phenyl-1,3,5-triazine-2,4-diyl)diimino]bisanthraquinone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

other: in vitro turn key strategy

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Oct 2014 - 02 Feb 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:

other: OECD (2014a) Draft Proposal for a New Test Guideline: Reconstructed Human Cornealike Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage

Principles of method if other than guideline:

The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol-extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control tissues and expressed as relative tissue viability.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-[(6-phenyl-1,3,5-triazine-2,4-diyl)diimino]bisanthraquinone

EC Number:

223-912-2

EC Name:

1,1'-[(6-phenyl-1,3,5-triazine-2,4-diyl)diimino]bisanthraquinone

Cas Number:

4118-16-5

Molecular formula:

C37H21N5O4

IUPAC Name:

1,1'-[(6-phenyl-1,3,5-triazine-2,4-diyl)diimino]di(9,10-anthraquinone)

Details on test material:

- Physical state: Solid / yellow
- Storage condition of test material: Room temperature

Test animals / tissue source

Species:

other: reconstructed three dimensional human cornea model

Strain:

other: Tissue model: OCL-200

Details on test animals or tissues and environmental conditions:

The EpiOcular™ 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). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on especially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control (NC): De-ionized water, sterile Positive control (PC): Methyl acetate (98+%, CAS No.: 79-20-9)

Amount / concentration applied:

TEST MATERIAL
The test substance is applied undiluted; therefore no preparation of the test substance in a vehicle was performed.

Duration of treatment / exposure:

After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

Number of animals or in vitro replicates:

Two tissues were treated with each, the test substance, the PC and the NC.

Details on study design:

PREINCUBATION OF THE TISSUES
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 preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

PRETREATMENT OF THE TISSUES
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.

APPLICATION OF THE TEST SUBSTANCE
Using a sharp spoon, a bulk volume of 50 μL of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

REMOVAL OF TEST SUBSTANCE
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 prewarmed
medium (post-soak immersion) in order to remove residual test substance. After 25 minutes (solids) 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).

Results and discussion

In vitro

Results

Irritation parameter:

other: Tissue viability in %

Remarks:

average of 2 samples

Run / experiment:

#1

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

In vivo

Other effects:

The test substance is not able to reduce MTT directly.

Any other information on results incl. tables

RESULTS OF THE EPIOCULAR TEST

Test substance		tissue 1	tissue 2	mean	inter-tissue variability [%]
NC	mean OD570	1.555	1.424	1.490
	viability [% of NC]	104.4	95.6	100	8.8
14/0579-1	mean OD570	1.687	1.410	1.548
	viability [% of NC]	113.2	94.7	104	18.6
PC	mean OD570	0.400	0.363	0.382
	viability [% of NC]	26.9	24.4	26	2.5

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results for the EpiOcular Test it was concluded, that the test article does not show an eye irritation potential under the test conditions chosen.

Executive summary:

The potential of the test article to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 20 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by a 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 104%.