Disodium 2-[4-[[2-cyano-3-[4-[methyl(2-sulphonatoethyl)amino]phenyl]-1-oxoallyl]amino]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Description of key information

Skin irritation: The skin irritation potential of the test substance was tested in an OECD guideline in-vitro study. No irritation potential was found. 
Eye irritation: The eye irritation potential of the test substance was tested in an OECD guideline in-vitro studiy. No eye irritation potential was found. 

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

Study period:

2015-05-04 to 2015-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia containing: 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® 1 cm

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

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 and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: No control animals because Human skin model. Negative and positive control were used in this test.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 25 µL

Duration of treatment / exposure:

1 hour

Observation period:

not applicable

Number of animals:

The irritation test was performed with three tissue samples

Details on study design:

Treatment
Time: 60 min
Incubator: Within this period the 6-well plates were put into the incubator at 37 °C.
Rinsing: After the end of the treatment interval the inserts were removed with PBS (Dulbecco's Phosphate Buffered Saline)

MTT Assay
A volume of 300 µL of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution was added to each well after the post-incubation period and the tissues were incubated.
Incubation period: 3 hours
Time: After the 3 hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT plates.
Rinsing: After 3 h incubation, wells were washed with PBS.
Extractant solution: Isopropanol

OD reading: SunriseTM Absorbance Reader, For the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter.

Irritation / corrosion parameter:

other: other: relative absorbance (% of negative control)

Value:

89.6

Remarks on result:

other:

Remarks:

Basis: other: test item, mean value of 3 tissues. Time point: 60 min. Max. score: 100.0. Reversibility: other: not examined. (migrated information)

Irritation / corrosion parameter:

other: other: relative absorbance (% of negative control)

Value:

2.8

Remarks on result:

other:

Remarks:

Basis: other: positive control, mean value of 3 tissues. Time point: 60 min. Max. score: 100.0. Reversibility: other: not examined. (migrated information)

Irritant / corrosive response data:

In tissues that have been treated with the test substance, a yellow discoloration was noticed after the washing procedure. Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 4.4 % of NC). Thus for the test substance the final mean viability is given after KC correction. The results of the KC tissues showed high inter-tissue variability. However, taking the highest single value of the test-substance treated KC tissues into account (tissue 2 KC: 12.5% of NC) the final result still corresponds to a viability value well above the cut off for skin irritation (about 82%), thus this deviation is not considered to adversely affect the outcome of this study.

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

Study period:

2015-04-13 to 2015-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: 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

Strain:

other: not applicable

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. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on especially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Vehicle:

unchanged (no vehicle)

Controls:

other: No control animals because Human skin model. Negative and positive control were used in this test.

Amount / concentration applied:

50 µL of the test item

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

other: 2 EpiOcular tissue samples were incubated with the test substance

Details on study design:

Treatment
Time: 6 hours
Rinsing: At the end of the treatment time, the test item was removed by rinsing the tissues 3 times in each of three beakers filled with PBS (Dulbecco’s Phosphate Buffered Saline).

MTT Assay
A volume of 300 µL of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide solution) was added.
Incubation period: 3 hours
Extractant solution: Isopropanol

OD reading: The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically.

Irritation parameter:

other: relative absorbance (% of negative control)

Basis:

other: test substance, mean of 2 tissues

Time point:

other: 6 hours

Score:

78.7

Max. score:

100

Reversibility:

other: not examined

Irritation parameter:

other: relative absorbance (% of negative control)

Basis:

other: positive control, mean of 2 tissues

Time point:

other: 6 hours

Score:

29.4

Max. score:

100

Reversibility:

other: not examined

Irritant / corrosive response data:

In tissues that have been treated with the test substance, a yellowish discoloration was noticed after the washing procedure. Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 10.7 % of NC). Thus for the test substance the final mean viability is given after KC correction.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Key

The objective was to assess the potential for corrosive activity and skin irritation of the test item. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT). However, in the current case for test item the results derived with SIT alone were sufficient for a final assessment. Therefore further testing in SCT was waived. The potential of the test item to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (ca. 18 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissue samples, 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin irritation test showed the following results: The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control (freeze-killed control tissues (KC)) 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 89.6%. In tissues that have been treated with the test substance, a yellow discoloration was noticed after the washing procedure. It was concluded, that the test item does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Disregarded

An aqueous solution of test item (10.8 g/100 g active ingredient) was assessed in a study according to OECD guideline 404 by a single topical application of 0.5 mL of the test substance to the intact skin of 3 White New Zealand rabbits for 4 hours under semiocclusive dressing. The average score (24 to 72 hours) for irritation was calculated to be 1.0 for erythema and 0.0 for edema. The skin findings were reversible in all animals within 7 days after removal .of the patches; thus the study was terminated. Under the test conditions chosen and considering the described findings the test material did not give indication of an irritant property to the skin (BASF, 1998). The study was rated as disregarded study as it was only tested in a purity of 10.8 g/100 g active ingredient.

Eye irritation:

Key

The objective of the present study was the determination of a possible eye irritating potential of the test item. The potential of the test item to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (ca. 26 mg) of the undiluted test substance 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. 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The EpiOcular™ eye irritation test showed the following results: The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced. The mean viability of the test-substance treated tissues was 78.7%. In tissues that have been treated with the test substance, a yellowish discoloration was noticed after the washing procedure. Based on the observed results for the EpiOcular Test it was concluded, that the test item does not show an eye irritation potential under the test conditions chosen.

Disregarded

An aqueous solution of test item (10.8 g/100 g active ingredient) was assessed in a study according to OECD guideline 405 in 3 White New Zealand rabbits, subjected to a single ocular application of 0.1 mL of the test material. The average score (24 to 72 hours) for irritation was calculated to be 0.0 for coneal opacity, for iris and for chemosis and 0.3 for conjunctivae redness. The findings were reversible in all animals within 72 hours after application; thus the study was terminated. Under the test conditions chosen and considering the described findings the test material did not give indication of an irritant property to the eye (BASF, 1998). The study was rated as disregarded study as it was only tested in a purity of 10.8g/100 g active ingredient.

Justification for selection of skin irritation / corrosion endpoint:
GLP and guideline study

Justification for selection of eye irritation endpoint:
GLP and guideline study

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin and eye irritation under Regulation (EC) No 1272/2008.