Cobaltate(3-), bis[6-amino-5-[(2-hydroxy-3,5-dinitrophenyl)azo]-1-naphthalenesulfonato(3-)]-, sodium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read across justification is detailed in section 13; source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

The results derived with EpiOcular alone were sufficient for a final assessment. Therefore further testing in the BCOP was waived.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline (EpiOcularTM)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcularTM model (OCL-200) is a 3D non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on especially prepared 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
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: killed tissue control

Amount / concentration applied:

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) or test substance (killed tissue control, KC).

Duration of treatment / exposure:

Incubation for 6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2 tissues treated with each, test substance, PC and NC.
2 killed tissues used for each, the test substance and the NC.

Details on study design:

EXPERIMENTAL PROCEDURE
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest 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 55 to 65 minutes. 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.
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 substance in the same way as the viable tissues.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 54) 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.

Pre-incubation 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) or test substance (killed tissue control, KC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

Removal of the test substance and postincubation period
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).

MTT incubation
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.


DATA EVALUATION
The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material was an irritant.

Calculation of individual and mean optical densities
The corrected measured OD570 value for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated.

Application of measurements using killed control tissues
In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) was used to correct the mean OD570 of the test substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC was calculated by subtracting the mean OD570 KC of the NC from the mean OD570 KC of the test substance. In case the mean net OD570 KC was greater than 0.1, it was subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance. In case the mean net OD570 KC was smaller than 0.1, the effect of direct MTT reduction was considered to be negligible and no subtraction followed.

Tissue viability
The quantification of tissue viability is presented as the ratio of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.

Assay acceptance criterion for the NC
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Acceptance criteria for the PC
Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 60% is acceptable.

Assay acceptance criterion for tissue variability
Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the relative difference of the viability is ≤ 20%.

Acceptance criteria for the KC
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTT-reduction of a test substance should be ≤ 50% of the OD570 of the NC.


EVALUATION OF RESULTS
The evaluation of the eye irritation potential of the test substance uses the results of the BCOP Test and the EpiOcular Test.
If a test substance was not tested in both of the test systems or an inconclusive result was obtained in one of the tests, the test strategy might still lead to an overall evaluation when the other test system result gives a clear prediction. However, if contradictory results are obtained a test evaluation might not be possible.

Evaluation of results BCOP
The following decision criteria apply: IVIS
≤ 3 no classification for eye irritation
> 3; ≤ 55 no prediction can be made for eye irritation, further testing with another suitable method is required
> 55 ocular corrosive or severe irritant

Evaluation of results EpiOcular
The following decision criteria apply: mean tissue viability (% of negative control)
≤ 60 prediction: irritant
> 60 prediction: non-irritant

Combined assessment of eye irritating potential
If in a top-down approach, the BCOP test resulted in the prediction of ocular corrosive or severe irritant, no EpiOcular was conducted. If in a bottom-up approach EpiOcular resulted in non-irritant, no BCOP test was conducted. In all other cases both assays were necessary for a final assessment.
The following criteria apply:
BCOP result: ocular corrosive or severe irritant and EpiOcular result: irritant => overall result: ocular corrosive or severe irritant
BCOP result: not identified as corrosive or severe irritant and EpiOcular result: irritant => overall result: irritant
BCOP result: not identified as corrosive or severe irritant and EpiOcular result:non-irritant => overall result: non-irritant

Results and discussion

In vitro

Results

Irritation parameter:

other: viability (% of NC)

Run / experiment:

EpiOcular test

Value:

78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

In vivo

Other effects:

Due to the intense color of the test substance it could not be determined whether the test substance is able to reduce MTT directly. Therefore an additional MTT reduction control was introduced. The result of the control tissues inactivated by freezing (KC) indicated an increased MTT reduction.
Compound residues remained on the tissues after the washing procedure. However, this did not interfere with the colorimetric test as was demonstrated in a pretest.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Based on the observed results for the EpiOcular™ Test alone and applying the evaluation criteria it was concluded, that test substance does not show an eye irritation potential under test conditions chosen.

Executive summary:

Method

Two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, results on test substance derived with EpiOcular alone were sufficient for a final assessment. Therefore further testing in BCOP was waived.

The potential of test substance to cause ocular irritation was assessed by a single topical application of 50 μl bulk volume (about 15 mg) of the undiluted test substance to a reconstructed 3D 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 test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

Results

Due to the intense color of the test substance it could not be determined whether the test substance is able to reduce MTT directly. Therefore an additional MTT reduction control was introduced. The result of the control tissues inactivated by freezing (KC) indicated an increased MTT reduction.

Compound residues remained on the tissues after the washing procedure. However, this did not interfere with the colorimetric test as was demonstrated in a pretest.

The final mean viability of the test-substance treated tissues was 78%.