Reaction mass of 5,5'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[3-methylsalicylic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and potassium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxy-7-sulfonaphthalene-2-sulfonate - 2,2',2''-nitrilotriethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol

Endpoint summary

Description of key information

The test substance does not show a skin irritation potential in the EpiDerm in vitro skin test.

The test substance does not show an eye irritation potential in the in vitro eye irritation test.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

Three dimensional human epidermis model
The EpiDermTM 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: EPI-200
Tissue Lot Number: 23377 (test run 1) and 23385 (test run 2) (Certificates of Analysis see appendix)
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Irritation test:
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 control tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction.
25 µL sterile PBS was applied first. As the test substance could not be applied with a sharp spoon, a metal pin was covered with a bulk volume of ca. 25 µL of the solid test material and was applied with direct contact to the tissue.
Control tissues were concurrently treated with 30 µL of sterile PBS (NC, NC 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 placed into 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 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. 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.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 25µl bulk volume (about 44 mg).

Irritation / corrosion parameter:

% tissue viability

Value:

92

Irritation test: 2nd test run

Test substance identification			tissue 1	tissue 2	tissue 3	mean	SD	CV[%]
NC	viable tissues	mean OD570	1.980	1.997	1.873
		viability [% of NC]	101.5	102.4	96.1	100.0	3.4	3.4
	KC tissues	mean OD570	0.045	0.049	0.048	0.047
		viability [% of NC]	2.3	2.5	2.5	2.4	0.1	5.0
16/0255-1	viable tissues	mean OD570	1.742	1.760	1.888	1.797
		viability [% of NC]	89.3	90.2	96.8	92.1	4.4	4.4
	KC tissues	mean OD570	0.000	0.000	0.006	0.002
		viability [% of NC]	0.0	0.0	0.3	0.1	0.2	173.2
	Final mean viability of tissues after KC correction[% of NC]:					92.0
PC	viable tissues	mean OD570	0.047	0.046	0.052	0.049
		viability [% of NC]	2.4	2.4	2.7	2.5	0.2	6.6

* Negative values are set to zero for further calculation

The tissues were black discolored and compound 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 0.1% of NC). Thus for the test substance the final mean viability is given after KC correction.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results and applying the evaluation criteria, it was concluded, that Direct Black 18L NA active dye does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

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 (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs®, 10 mm ø) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 23747 (Certificate of Analysis see apendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):50 µl bulk volume (about 70 mg)

Details on study design:

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. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.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.
The color of a test substance may interfere with the color density produced by metabolic capacity of the tissue and would falsify the test results when residues of the test substance remain on the tissues after washing and are extracted by the isopropanol. 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. 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.
Several test substances were tested in parallel within the present test (test no. 81) using the same control tissues (NC and PC).
Two tissues were treated with each, the test substance, the PC and the NC. In addition two killed tissues were used for each, the test substance and the NC, in order to detect direct MTT reduction. There are two separate protocols for liquids and solids, differing in exposure time and post-incubation period. Due to the physical state of the test substance the protocol for solids was applied. 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. The test substance could not be applied with a sharp spoon. Therefore a metal pin was covered with a bulk volume of ca. 50 µL of the undiluted test material and was applied with direct contact to the tissue. 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). 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.
Negative control (NC): De-ionized water, sterile
Positive control (PC): Neat methyl acetate (CAS No.:79-20-9)

Remarks on result:

other: The mean viability o fthe test-substance treated tissues was 80.8%.

Test substance identification			tissue 1	tissue 2	mean	Inter-tissuee variability[%]
NC	viable tissues	mean OD570	1.506	1.469	1.487
		viability [% of NC]	101.2	98.8	100.0	2.5
	KC tissues	mean OD570	0.033	0.025	0.029
		viability [% of NC]	2.2	1.7	2.0	0.5
16/0255-1	viable tissues	mean OD570	1.282	1.32	1.207
		viability [% of NC]	86.2	76.1	81.2	10.1
	KC tissues	mean OD570	0.004	0.008	0.006
		viability [% of NC]	0.3	0.5	0.4	0.2
	Final mean viability of tissues after KC correction[% of NC]:				80.8
PC	viable tissues	mean OD570	0.152	0.209	0.181
		viability [% of NC]	10.2	14.1	12.2	3.8

 

The tissues were black discolored and compound 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 0.4% of NC). Thus for the test substance the final mean viability is given after KC correction.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that Direct Black 18L NA active dye does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The objective was to assess the skin irritation and corrosion potential of Direct Black 18L NA active dye. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT). However, in the current case for Direct Black 18L NA active dye the results derived with SIT alone were sufficient for a final assessment. Therefore, further testing in SCT was waived. The potential of Direct Black 18L NA active dye to cause dermal irritation was assessed by a single topical application of ca. 25 µL bulk volume (about 44 mg) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). The test substance could not be applied with a sharp spoon. Therefore, a metal pin was covered with a bulk volume of ca. 25 µL of the undiluted test material and was applied with direct contact to the tissue. The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin irritation test: Due to the color of the test substance it could not be determined whether the test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control was introduced. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 55.4%. Due to non-concordant replicate measurements of the test-substance treated tissues (values for single tissues: 13.7%, 108.8% and 45.3%), another test run was performed to clarify this result. In the 2^ndtest run the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 92.0%. All acceptance criteria were met. In both tests the tissues were black discolored and compound residues remained on the tissues after the washing procedure. However, this did not interfere with the colorimetric test as was demonstrated in a pretest. Based on the observed results and applying the evaluation criteria, it was concluded, that Direct Black 18L NA active dye does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye Irritation:

The objective was to assess the eye irritating potential of Direct Black 18L NA active dye. Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test. However, in the current case for Direct Black 18L NA active dye the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived. The potential of Direct Black 18L NA active dye to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (about 70 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™). The test substance could not be applied with a sharp spoon. Therefore, a metal pin was covered with a bulk volume of ca. 50 µL of the undiluted test material and was applied with direct contact to the tissue. Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular™ eye irritation assay: Due to the color of the test substance it could not be determined whether the test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control was introduced. The tissues were black discolored and compound residues remained on the tissues after the washing procedure. However, this did not interfere with the colorimetric test as was demonstrated in a pretest. The mean viability of the test-substance treated tissues was 80.8%. Based on the observed results for the EpiOcular Test alone and applying the evaluation criteria it was concluded, that Direct Black 18L NA active dye does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

Based on the results, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).