Disodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

other: experimental data on similar substance

Adequacy of study:

key study

Study period:

2016-11-30 to 2016-01-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

28 July 2015

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:

Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

EC Number:

276-770-9

EC Name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Cas Number:

72705-24-9

Molecular formula:

C26H24N4O9S3.H3N.Na

IUPAC Name:

ammonium sodium 2-[4-({1-[(2-methoxy-5-methyl-4-sulfonatophenyl)carbamoyl]-2-oxopropyl}diazenyl)phenyl]-6-methyl-1,3-benzothiazole-7-sulfonate

In vitro test system

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

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

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C ± 1°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1x after 1 hour application
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 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.

NEGATIVE CONTROL
- Amount applied: 30 μL of sterile Dulbecco's phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount applied: 30 μL of SDS
- Concentration: 5%

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

After 24 ± 2 hours for additional 18 ± 2 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

97.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

125.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

90.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: Based on the results of the pretest it was judged, that application of color control tissues is not necessary.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Test substance
identification Tissue 1 Tissue 2 Tissue3 mean SD CV [%]
NC mean OD570 2.128 2.338 1.997 2.152
viability
[% of NC] 98.8 108.5 92.7 100.0 8.0 8.0
Test substance mean OD570 2.102 2.698 1.956 2.252
viability
[% of NC] 97.6 125.2 90.8 104.5 18.3 17.5
PC mean OD570 0.071 0.076 0.069 0.072
viability
[% of NC] 3.3 3.5 3.2 3.3 0.2 4.7

Any other information on results incl. tables

Table 1: Decision criteria for evaluation of results of irritation test

Mean tissue viability (% of negative control)	Prediction
< 45	Irritant
45 - 55	Borderline
> 55	Non-irritant

The „borderline“-evaluation (50 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.

Table 2: Results

Test substance identification		Tissue 1	Tissue 2	Tissue 3	mean	SD	CV [%]
NC	mean OD570	2.128	2.338	1.997	2.152
	viability [% of NC]	98.8	108.5	92.7	100.0	8.0	8.0
Test substance	mean OD570	2.102	2.698	1.956	2.252
	viability [% of NC]	97.6	125.2	90.8	104.5	18.3	17.5
PC	mean OD570	0.071	0.076	0.069	0.072
	viability [% of NC]	3.3	3.5	3.2	3.3	0.2	4.7

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Not skin irritant