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

Skin irritation / corrosion

Administrative data

Endpoint:

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model 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
- Lot/batch No.: #0011852462
- Test-substance No.: 14/0416-1
- Content: About 90 g/100 g
- Storage condition of test material: Room temperature, dry storage, no direct sunlight
- Homogeneity: homogeneous by visual inspection
- Physical state / color: Solid / orange to red

Test animals

Species:

other: in vitro: reconstructed human epidermal model EpiDermTM

Details on test animals or test system and environmental conditions:

24 EPI-200 tissues (reconstructed epidermis) were obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
The EpiDerm™ model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Test system

Vehicle:

other: PBS

Controls:

other: negative control; PBS and positive control; 5% (w/v) sodium dodecyl sulfate in water

Amount / concentration applied:

ca. 25 µL bulk volume (about 24 mg) of the undiluted test substance.

Duration of treatment / exposure:

1 hours

Observation period:

42-hours post-incubation period

Number of animals:

Three 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°C ± 1°C, 5% ± 1% CO2, 90% ± 5% humidity.
- Tissue for MTT-reduction control: EPI-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).
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (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 minimally moistened with PBS.

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 cells.
- Basic procedure: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 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 for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. In addition three killed tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. 25 µL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 µL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently applied with 30 µL of sterile PBS (NC, NC, or KC) or with 30 µL of 5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue surface of the NC, NC, KC, and PC afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation 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. 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 quotient 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 is an irritant. The individual tissue OD570 is 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 three tissues treated in the same way is 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 quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not met repetition of the test is 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 value following exposure to Triton X-100 (1%) for each EpiDermTM 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 Guidelines: Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control 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 2.8.
- Acceptance criteria for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Acceptance criteria for tissue variability: For every treatment three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 18%.
- Acceptance criteria for killed controls (KC): The OD570 of the tissues for the KC of the NC should be ≤ 0.35. The OD570 value for direct MTT-reduction of a test substance should be ≤ 30% of the OD570 of the NC.

EVALUATION OF RESULTS
Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: mean tissue viability

Value:

89

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 h. Max. score: 100.0. Remarks: The mean tissue viability was 89.0 % after KC correction. (migrated information)

In vivo

Irritant / corrosive response data:

See any other information on results incl. tables

Other effects:

- Slight (viable tissues) or massive (KC tissues) compound-related discoloration of the tissues were observed 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 0.4% of NC). Thus for the test substance the final mean viability is given after KC correction.

Any other information on results incl. tables

Mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test Substance			mean	SD	CV [%]
NC	viable tissues	Mean OD570	2.151	0.011
		Viability [% of NC]	100.0	0.5	0.5
	KC tissues	Mean OD570	0.058	0.002
		Viability [% of NC]	2.7	0.1	3.0
14/0416-1	viable tissues	Mean OD570	1.923	0.099
		Viability [% of NC]	89.4	4.6	5.1
	KC tissues	Mean OD570 KC NC corrected	0.008	0.003
		Viability [% of NC]	0.4	0.1	36.9
	Final mean viability of tissues after KC correction [% of NC]		89.0 (%)
pc	viable tissues	Mean OD570	0.080	0.010
		Viability [% of NC]	3.7	0.5	13.0

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information