2-[4-[(hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-02-25 to 2016-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

IUPAC Name:

Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Specific details on test material used for the study:

Batch no.: 0013479406

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
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.

- Description of the cell system used
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 (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
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.

- RhCE tissue construct used, including batch number
Tissue model: OCL-200
Tissue Lot Number: 23700
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Doses of test chemical and control substances used
Test chemical: Undiluted
Negative control (NC): De-ionized water, sterile
Positive control (PC): neat

- Temperature of exposure/ incubation: 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) 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 3 hours. A negative control (de-ionized water) was tested concurrently. The test substance is not able to reduce MTT directly.

Color control
Due to the color of the test substance a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed as follows: the test substance was applied to a KC tissue, incubated and removed by washing in the same way as in the main experiment.
Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically.
Based on the result of the pretest, it was judged that application of color control tissues is not necessary.

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

- Wavelength used for quantifying MTT formazan
Spectrophotometer: SunriseTM Absorbance Reader
Wavelength: 570 nm (OD570)

- Description of the method used to quantify MTT formazan
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.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model

Decision criteria for evaluation of results
Mean tissue viability (% of negative control)
< 55%: irritant
55 - 65 %: Borderline
> 65%: Non-irritant

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
Acceptance criteria 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 > 0.8. Them 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 < 50% is acceptable.

- Complete supporting information for the specific RhCE tissue construct used: Certificate of Analysis on test system quality is provided by the supplier (MatTek Corporation)

- Positive and negative control means and acceptance ranges based on historical data
Protocol for solids Jan 2014 - Feb 2016
Negative control: Mean OD: 1.674 +/- 0.164
Positive control: Mean OD: 0.403 +/- 0.092
Mean % viability: 24.0 +/- 4.7

- Acceptable variability between tissue replicates for positive and negative controls: < 20%.
- Acceptable variability between tissue replicates for the test chemical: < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

other: tissue viability [%]

Run / experiment:

Mean value of tissue 1and tissue 2

Value:

68.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

22.3 %

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met