Disodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

other: experimental data on similar substance

Adequacy of study:

key study

Study period:

2 Dec 2015 to 05 Jan 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

EC Number:

276-770-9

EC Name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Cas Number:

72705-24-9

Molecular formula:

C26H24N4O9S3.H3N.Na

IUPAC Name:

ammonium sodium 2-[4-({1-[(2-methoxy-5-methyl-4-sulfonatophenyl)carbamoyl]-2-oxopropyl}diazenyl)phenyl]-6-methyl-1,3-benzothiazole-7-sulfonate

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Details on study design:

- Details of the test procedure used:
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.

- RhCE tissue construct used:
Tissue model: EpiOcularTM model (OCL-200)
Tissue Lot Number: 21588
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Duration:
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.
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 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.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan, and linearity range of measuring device (spectrophotometer): 570 nm (OD570), blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- Description of the method used to quantify MTT formazan
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.
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.
Tissue viability
The quantification of tissue viability is presented as the ratio of the mean OD570 divided by the respective OD570 NC value in percent.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
see in section "any other information"

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Yes

- Acceptable variability between tissue replicates for positive (PC) and negative controls (NC)
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 > 0.8. The mean OD570 of the NC should not exceed 2.5.
PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.

- Acceptable variability between tissue replicates for the test chemical
Two tissues were treated under the same conditions. A variability between the the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.

- Color control: Based on the results of the pretest it was judged, that application of color control tissues is not necessary.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other:

Remarks:

viability [% of NC]

Run / experiment:

tissue 1

Value:

66.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: viability [% of NC]

Run / experiment:

tissue 2

Value:

68.6

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

Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

 

Test substance identification		tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	mean OD570	1.652	1.514	1.583
	viability [% of NC]	104.4	95.6	100.0	8.7
Test substance	mean OD570	1.059	1.086	1.072
	viability [% of NC]	66.9	68.6	67.7	1.7
PC	mean OD570	0.479	0.584	0.532
	viability [% of NC]	30.2	36.9	33.6	6.7

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Not eye irritant