Reaction mass of aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and 5,12-dihydro-quino[2,3-b]acridine-7,14-dione

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

Remarks:

according to OECD and GLP

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

Reference substance name:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

EC Number:

278-207-2

EC Name:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

Cas Number:

75431-69-5

Molecular formula:

C20H12N2O5S.1/3Al

IUPAC Name:

aluminum tris(7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridine-2-sulfonate)

Constituent 2

Reference substance name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

213-879-2

EC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

1047-16-1

Molecular formula:

C20H12N2O2

IUPAC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Constituent 3

Reference substance name:

Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

EC Number:

243-319-2

EC Name:

Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Cas Number:

19795-24-5

Molecular formula:

C20H12N2O8S2.2/3Al

IUPAC Name:

dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Test animals / tissue source

Species:

other: human cornea model

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL bulk volume (~ 25 mg)

Duration of treatment / exposure:

6 hours followed by a 18 hours post-incubation period.

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

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. The EpiOcularTM tissues (surface 0.6 cm2) are cultured on cell culture inserts (MILLICELLsR, diameter 10 mm) and are commercially available as kits (EpiOcularTM 200), containing 24 tissues on shipping agarose.

Tissue model: OCL-200
Lot number: 23747
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after short 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 tissue the induced cytotoxicity (= loss of viability) is measured in a colometric 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 densitiy of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control (NC) tissues and expressed as relative tissue viability.

On the day of arrival 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 at standard culture conditions for 16 - 24 hours.
After pre-incubation the tissues were pre-treated with 20 µL DPBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Two tissues were treated with the test substance, the positive and negative control respectively. In addition two killed tissues were used for the test substance and the negative control respectively in order to detect direct MTT reduction.
A bulk volume of 50 µL of the test substance was applied covering the whole tissue surface.
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). Then, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.
The tissues were washed with sterile DPBS to remove residual test material. For this purpose the tissues were immersed and swiveled three times in aeach of three beakers filled with DPBS.
Rinsed tissues were immediately immersed into new 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 post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated for 3 hours. Thereafter the tissues were washed with DPBS 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 for at least 2 hours on a plate shaker.
The optical densitiy at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol fro each microtiter plate.
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test substance and the mean OD570 values of the NC is used for evaluating whether a test substance is irritant or not irritant.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Run / experiment:

mean viability of tissues after KC correction

Value:

96.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Findings:

substance			tissue 1	tissue 2	mean	Inter-tissue variability [%]
negative control	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
positive control	viable tissues	mean OD570	0.152	0.209	0.181
		viability [% of NC]	10.2	14.1	12.2	3.8
test substance	viable tissues	mean OD570	1.410	1.478	1.444
		viability [% of NC]	94.8	99.4	97.1	4.6
	KC tissues	mean OD570 KC NC corrected	0.007	0.002	0.004
		viability [% of NC]	0.2	0.4	0.3	45.1
	final mean viability of tissues after KC correction [% of NC]				96.8

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.3 % NC). thus for the test substance the final mean viability is given after KC correction.

The data show, that a treatment with the test item did not significantly affect the viability of tissues (relative viabilty = 96.8 %)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Regulation (EC) 1272/2008

Conclusions:

The test substance did not show an eye irritation potential in the EpiOcularTM in vitro eye irritation test. Therefore the substance has not to be classified according to Regulation (EC) 1272/2008.

Executive summary:

The potential of the test substance to cause ocular irritaion was assessed by single topical application of ca. 50 µL bulk volume (about 25 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular^TM).

Two EpiOcular^TMtissues 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 ot mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues was compared to that of negative control tissues. The ratio of both values indicated the realtive tissue viability.

The following test results were obtained in the EpiOcular^TM eye irritation test:

The test substance was able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

In a pre-test it was demonstraed that the color of the test substance did not interfere with the colorimetric test.

The mean viability of the test substance treated tissues was 96.8 %. All acceptance criteria were met.

The test substance does not show an eye irritation potential in the EpiOcular^TMin vitro eye irritation test. Therefore the substance has not to be classified according to Regulation (EC) 1272/2008.