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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

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)

Version / remarks:

2014

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

219-694-3

EC Name:

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

Cas Number:

2498-95-5

Molecular formula:

C27H24N4O7S3.2Na

IUPAC Name:

disodium 2-{4-[(2-cyano-3-{4-[methyl(2-sulfonatoethyl)amino]phenyl}acryloyl)amino]phenyl}-6-methyl-1,3-benzothiazole-7-sulfonate

Test animals

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.

Test system

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.

Results and discussion

In vitro

Results

Results 1

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)

Results 2

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)

In vivo

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.