Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (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:

2017-01-03 to 2017-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

304-519-6

EC Name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

94276-33-2

Molecular formula:

C16 H12 Cr N4 O9 S . C11 H25 N O . H

IUPAC Name:

hydrogen 3-[(2-ethylhexyl)oxy]propan-1-amine 11-methyl-4-nitro-12-(phenylcarbamoyl)-6-sulfonato-8λ³-oxa-10λ³-oxa-1λ⁴,13-diaza-9-chromatricyclo[7.4.0.0²,⁷]trideca-1(13),2(7),3,5,10-pentaene-9,9,9-tris(ylium)-8,12-diid-9-olate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 002-152503

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST
20 % (w/v) suspension in de-ionized water

OTHER SPECIFICS: Solid / orange to brown, pH ca. 5 (undiluted test substance moistened with deionized water or 20 % aqueous preparation, determined in the lab prior to start of the GLP study)

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
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 (bulk volume)

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:
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 pre-incubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours. 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.

- RhCE tissue construct used, including batch number:
OCL-200, Lot No 23760, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Doses of test chemical and control substances used:
Using a sharp spoon, a bulk volume of ca. 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).

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
exposure: 6 h, 37 °C
post exposure immersion: 25 min, room temperature
post-exposure incubation: 18 h, 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
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. 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 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 for 6 hours and removed by washing. 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, and linearity range of measuring device: SunriseTM Absorbance Reader, 570 nm

- 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
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.


- Complete supporting information for the specific RhCE tissue construct used
see under "Any other information on materials and methods"

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability (%)

Value:

106

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No, ninimal yellowish discoloration of the test-substance treated tissues was observed after the washing procedure

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Table 4: 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	1.490	1.523	1.506
	Viability [% of NC]	98.9	101.1	100.0	2.2
Test substance	Mean OD570	1.794	1.401	1.597
	Viability [% of NC]	119.1	93.0	106.0	26.1
PC	Mean OD570	0.345	0.222	0.283
	Viability [% of NC]	22.9	14.7	18.8	8.2

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met