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

Endpoint summary

Description of key information

The test substance is not considered to be a skin or eye irritant in vitro and in vivo.

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:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

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

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

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.

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%.

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)

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.

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

Interpretation of results:

not irritating

Remarks:

Migrated information

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:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

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

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.

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%.

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.

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

Interpretation of results:

not irritating

Remarks:

Migrated information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion

An in vitro skin irritation test was conducted according to OECD 439 and in compliance with GLP. Ca. 25 µL bulk volume (about 24 mg) of the undiluted test substance was applied to a reconstructed three dimensional human epidermis model (EpiDerm™).Three EpiDerm™ tissue samples 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. Due to the intense 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 (freeze-killed control tissues (KC)) was introduced. Compound-related discoloration of the tissues was noticed 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 determined after an exposure period of 1 hour with about 42 hours post-incubation was 89%. Under the conditions of the test it was concluded that the test substance does not show skin irritation potential.

 

In an OECD 404 guideline study, the potential to cause acute dermal irritation or corrosion was assessed by a single topical application of 0.5 mL of the undiluted substance (containing 20% of the active ingredient). Three New Zealand White rabbits were exposed for 4 hours, using a patch of 12-16 cm, covered with a semi-occlusive dressing. The cutaneous reactions were assessed immediately after removal of the patch and approximately 24, 48 and 72 hours later. As no skin irritation was observed from 24 to 72 hours the test wat terminated after 72 hours evaluation. Both the erythema score as the edema score were determined to be 0 for all exposed animals at all time points. The substance was not considered to be a skin irritant under the conditions of this test.

 

Eye irritation

An in vitro eye irritation test was performed, with the reaction mass, according to OECD 492 and in compliance with GLP. Ca. 50 µL bulk volume (about 26 mg) of the undiluted test substance was applied to a reconstructed three dimensional human cornea model (EpiOcular™).Two EpiOcular™ tissue samples 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. Due to the intense 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 (freeze-killed control tissues (KC)) was introduced. Test substance 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 77.4%. Under the condition of the test it was concluded that the test substance does not show eye irritation potential.

 

In an OECD 405 guideline study, the potential to cause damage to the conjunctiva, iris or cornea was assessed in three New Zealand White rabbits subjected to a single ocular application of 0.1 mL of the test substance (containing 20% of the active ingredient) on day 0. The mean score (24 to 72 hours) for irritation was calculated to be 0 for corneal opacity, iritis, conjunctival redness and chemosis. The substance was therefore not considered to be irritating to the eyes. 

 

Justification for selection of skin irritation / corrosion endpoint:
There are two studies available. The study performed with the highest concentration active ingredient (90%) was selected as key.

Justification for selection of eye irritation endpoint:
There are two studies available. The study performed with the highest concentration active ingredient (90%) was selected as key.

Justification for classification or non-classification

No skin or eye effects were observed in animals exposed to the test substance in in vitro assays. In addition, in vivo tests with 20% of the active ingredient also showed no signs of skin and eye irritating potential. Based on this information classification for skin and eye irritation is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.