Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]benzenesulphonamidato(2-)]cobaltate(1-)

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:

other: GLP guideline study

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:

Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]benzenesulphonamidato(2-)]cobaltate(1-)

EC Number:

261-204-5

EC Name:

Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]benzenesulphonamidato(2-)]cobaltate(1-)

Cas Number:

58302-43-5

Molecular formula:

C32H22CoN6O8S2.Na

IUPAC Name:

sodium [4-(hydroxy-kappaO)-3-{[2-(hydroxy-kappaO)-1-naphthyl]diazenyl}benzenesulfonamidato(2-)][4-hydroxy-3-{[2-(hydroxy-kappaO)-1-naphthyl]diazenyl}benzenesulfonamidato(2-)]cobaltate(1-)

Details on test material:

- Name of test material (as cited in study report): Eukesolar Rubine EB Liquid - dried
- Physical state: Solid / dark red
- Analytical purity:
HPLC fingerprint:
87.0 area-% (268 nm) sum of all peaks: 99.4 area-%
90.1 area-% (552 nm) sum of all peaks: 100.2 area-%
(For details see analytical report No.: 14L00142)
- Lot/batch No.: Dye powder sample 14/076 from Material no. 52631478; batch no. M-R/G
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals / tissue source

Species:

other: not applicable

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

not applicable

Test system

Vehicle:

other: not applicable

Controls:

other: not applicable

Amount / concentration applied:

not applicable

Duration of treatment / exposure:

not applicable

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

OBJECTIVES
The objective of the present study is the determination of a possible eye irritating potential of
Eukesolar Rubine EB Liquid; dried. Using the currently available methods a single in vitro
assay may not always be sufficient to cover the full range of eye irritating potential.
Therefore, two assays are part of this in vitro eye irritation test strategy: The Bovine Corneal
Opacity and Permeability Test (BCOP) and EpiOcular Eye Irritation Test.
However, in the current case for Eukesolar Rubine EB Liquid; dried the results derived
with EpiOcular alone were sufficient for a final assessment. Therefore further testing in BCOP
was waived.
BCOP:
The objective of this in vitro test is to assess the potential of the test substance to cause
serious eye damage. According to the current version of the OECD Guideline 437 (adopted
July 2013), also substances that do not require classification for eye irritation or serious eye
damage can be identified. However, due to insufficient predictivity the latter derivation is not
recommended by the test facility (see chapter 3.10).
The test method consists of a topical exposure of the test substance to the epithelial surface
of isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured
quantitatively as the amount of light transmission through the cornea. Permeability is
measured quantitatively as the amount of sodium fluorescein dye that passes across the full
thickness of the cornea. Both measurements are used to calculate an In Vitro Irritancy Score
(IVIS) of the test substance, which is used for the prediction of serious eye damage.
EpiOcular:
The objective of this in vitro test is to assess the eye irritation potential of the test substance
using the reconstructed human ocular model EpiOcularTM.
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 isopropanolextraction
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.

TEST SYSTEM
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 especially prepared cell culture inserts (MILLICELLs, 10 mm ∅) 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

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.
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 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.
There are two separate protocols for liquids and solids, differing in exposure time and postincubation
period. Due to the physical condition of the test substance the protocol for solids
was applied.

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
Table(s) of measured parameters presented in the report were produced using a computerbased
tabular calculation software. The mean and individual data were not always rounded
but the significant digits were produced by changing the display format. As a consequence,
calculation of mean values using the individual data presented in the report will, in some
instances, yield minor variations in value.
Principle
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 testsubstance
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 Prediction: no classification for eye irritation
> 3; ≤ 55 Prediction: no prediction can be made for eye irritation, further testing with another suitable method is required
> 55 Prediction: 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: RESULTS OF THE EPIOCULAR TEST [viability (% of NC)]

Run / experiment:

mean value 6 hours

Value:

78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

The final mean viability of the test-substance treated tissues was 78% after incubation with the test substance for 6 hours followed by a 18-hours post-incubation period.

Any other information on results incl. tables

The objective of the present study was the determination of a possible eye irritating potential of Eukesolar Rubine EB Liquid; dried. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of eye irritating potential.

Therefore, 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, in the current case for Eukesolar Rubine EB Liquid; dried the results derived with EpiOcular alone were sufficient for a final assessment. Therefore further testing in BCOP was waived.

The potential of Eukesolar Rubine EB Liquid; dried 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 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:

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%.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU