Reaction mass of tetrasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(2-methyl-4-sulphonatophenyl)azo]naphthalene-2-sulphonate] and tetrasodium 4-[[1-hydroxy-6-[[[[5-hydroxy-6-[(2-methyl-4-sulphonatophenyl)azo]-7-sulphonato-2-naphthyl]amino]carbonyl]amino]-3-sulphonato-2-naphthyl]azo]benzoate and tetrasodium 4,4'-[carbonylbis[imino(1-hydroxy-3-sulphonatonaphthalene-6,2-diyl)azo]]dibenzoate

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 492 EpiOcular Eye Irritation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Tetrasodium 4-[[1-hydroxy-6-[[[[5-hydroxy-6-[(2-methyl-4-sulphonatophenyl)azo]-7-sulphonato-2-naphthyl]amino]carbonyl]amino]-3-sulphonato-2-naphthyl]azo]benzoate, Tetrasodium 4,4'-[carbonylbis[imino(1-hydroxy-3-sulphonatonaphthalene-6,2-diyl)azo]]dibenzoate and Tetrasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(2-methyl-4-sulphonatophenyl)azo]naphthalene-2-sulphonate]

IUPAC Name:

Reaction mass of Tetrasodium 4-[[1-hydroxy-6-[[[[5-hydroxy-6-[(2-methyl-4-sulphonatophenyl)azo]-7-sulphonato-2-naphthyl]amino]carbonyl]amino]-3-sulphonato-2-naphthyl]azo]benzoate, Tetrasodium 4,4'-[carbonylbis[imino(1-hydroxy-3-sulphonatonaphthalene-6,2-diyl)azo]]dibenzoate and Tetrasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(2-methyl-4-sulphonatophenyl)azo]naphthalene-2-sulphonate]

Details on test material:

- Name of test material (as cited in study report): Direct Orange 102 similar
- Content: About 90 g/100 g
- Lot/batch No.: #0011852462
- Test substance No.: 14/0416-1
- Storage condition of test material: Room temperature, dry storage, no direct sunlight
- Homogeneity: The test substance was homogeneous by visual inspection.
- Physical state / color: Solid / orange to red

Test animals / tissue source

Species:

other: in vitro, reconstructed human Cornea-like Epithelium

Details on test animals or tissues and environmental conditions:

The EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm diameter ) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Test system

Vehicle:

water

Controls:

other: Negative control: De-ionized water, sterile. positive control: Neat methyl acetate (CAS No.: 79-20-9).

Amount / concentration applied:

ca. 50 µL bulk volume (about 26 mg) of the undiluted test substance

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

18-hours post-incubation

Number of animals or in vitro replicates:

Two tissues were tested in parallel

Details on study design:

MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220, incubation conditions: 37 ± 1°C, 5 ± 1% CO2, 90 ± 5% humidity
- Spectrophotometer: SunriseTM Absorbance Reader, for the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter
- Tissue for MTT-reduction control: OCL-200 tissue that is killed by freezing at –20°C
- Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany)
- Pre-treatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany), 1.0 mg / mL assay medium
- Extracting agent: Isopropanol p.a.

ANALYSES
No analysis of test-substance preparation was performed, because the test substance was applied undiluted.

EXPERIMENTAL PROCEDURE
- Direct MTT reduction: 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. 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 article and the negative control in the same way as the unfrozen tissues.
- 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 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.
- Application of the test substance: 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, NC KC) or with 50 µL of methyl acetate (PC) or test substance (KC). After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed. 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 pre-warmed 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. 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. 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. In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be 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 is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is 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. 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.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 µL of 0.3% Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline: Lower acceptance limit: ET50 = 12.2 min; Upper acceptance limit: ET50 = 37.5 min
- 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 ≥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.
- Acceptance criteria 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 ≤ 30% of the NC.

EVALUATION OF RESULTS
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60%.

Results and discussion

In vivo

Results

Irritation parameter:

other: mean tissue viability

Basis:

mean

Time point:

other: 6 h

Score:

77.4

Max. score:

100

Remarks on result:

other: The mean tissue viability was 77.4 %

Irritant / corrosive response data:

See any other information on results incl. tables.

Other effects:

- Minimal (viable tissues) to moderate (KC tissues) test substance residues remained on the tissues after the washing procedure.
- Due to the intense color of the test substance, the ability of the test substance to reduce MTT directly could not be determined in a pretest, therefore KC tissues were applied in parallel.
- The results of the KC tissues indicate an increased MTT reduction (mean viability: 0.7% of NC). Thus for the test substance the final mean viability is given after KC correction.

Any other information on results incl. tables

Mean OD₅₇₀values, individual and mean viability values and inter-tissue variability

Test Substance			mean	Inter-tissue variability [%]
NC	viable tissues	Mean OD570	1.793
		Viability [% of NC]	100.0	7.0
	KC tissues	Mean OD570	0.029
		Viability [% of NC]	1.6	0.0
14/0416-1	viable tissues	Mean OD570	1.400
		Viability [% of NC]	78.1	1.8
	KC tissues	Mean OD570 KC NC corrected	0.013
		Viability [% of NC]	0.7	0.4
	Final mean viability of tissues after KC correction [% of NC]		77.4%
pc	viable tissues	Mean OD570	0.434
		Viability [% of NC]	24.2	1.1

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information