Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato(2-)]chromate(1-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2015)

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato(2-)]chromate(1-)

EC Number:

276-067-7

EC Name:

Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato(2-)]chromate(1-)

Cas Number:

71839-81-1

Molecular formula:

C34H28Cl2CrN10O8S2.Na

IUPAC Name:

Chromate(1-), bis[3-[2-[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-(oxo-kO)-1H-pyrazol-4-yl]diazenyl-kN1]-4-(hydroxy-kO)-N-methylbenzenesulfonamidato(2-)]-, sodium (1:1)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Other name: Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-N-methylbenzene-1-sulphonamidato
(2-)]chromate(1-)
C.I. Index Solvent Orange 41
CAS-No.: 71839-81-1
EINECS / EC-No.: 276-067-7
Appearance: Red powder
Expiry Date: 06.01.2026

In vitro test system

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues

Duration of treatment / exposure:

60 minutes.

Test animals

Species:

other: reconstituted human epidermis model

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
25 mg (~ 39 mg/cm2)

NEGATIVE CONTROL
30 µL DPBS (MatTek) were used as negative control per tissue.

POSITIVE CONTROL
30 µL of a 5% SLS solution in deionized water (MatTek) were used a positive control per tissue.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

See table below

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

all

Value:

ca. 111.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean relative absorbance value of the test item, corresponding to the cell viability, was 111.7% after taking the correction factor gained in the above mentioned additional test with a viable tissue into consideration (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin. 

Any other information on results incl. tables

Results after treatment with the test item and the controls (exposure interval of 60 minutes):

Dose Group	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 wells blank corrected	Mean Absorbance  of 3 wells blank corrected	Mean Absorbance of 3 tissues after blank correction	Rel. Absorb. [%] Tissue 1,2,3
Blank Controls		0.036	0.037	0.036	0.036
Negative Control	1	1.578	1.549	1.558	1.562	1.525	1.811	84.2
	2	2.026	1.984	1.971	1.994	1.957		108.8
	3	2.024	1.971	1.965	1.986	1.950		107.7
Positive Control	1	0.098	0.097	0.097	0.097	0.061	0.078	3.4
	2	0.118	0.121	0.118	0.119	0.082		4.6
	3	0.125	0.126	0.126	0.126	0.089		4.9
Blank Test Item		0.038	0.038	0.037	0.038
Test Item	1	2.095	1.978	1.990	2.021	1.984	2.056	109.5
	2	2.132	2.124	2.132	2.130	2.092		115.5
	3	2.140	2.126	2.127	2.131	2.094		115.6

Dose Group	Tissue No.	Relative Standard Deviation [%]	Mean Relative Absorbance [% of Negative Control] **	Mean Absorbance Blank corrected viable tissue without MTT (Step 2)	Mean Relative Absorbance [% of Negative Control] after colour interference and MTT reduction correction
Blank Controls
Negative Control	1
	2	13.7	100.0
	3
Positive Controls	1
	2	19.0	4.3
	3
Blank Test Item
Test Item	1
	2	3.1	113.6	0.034	111.7
	3

*    relative absorbance per tissue [rounded values]

**   relative absorbance per treatment group [rounded values]

  The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water lead to a change in colour. Therefore an additional experiment for unspecific colour interference was performed and the result was used as correction factor for the evaluation of true tissue viability.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, was 111.7% after taking the correction factor gained in the above mentioned additional test with a viable tissue into consideration (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin. 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item passed the MTT- pre- test. Because of possible colour interference an additional test with a viable tissue was performed and the result was used as correction factor for data evaluation in the main experiment.

Approximately 25 mg of the test item were applied to each tissue, wetted with 25 µL of DPBS, and spread to match the surface of triplicate tissues.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissues each.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 43 hours the tissues were treated with the MTT solution for 3 hours following nearly 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item and the negative control in the main test were below 19% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%). The slight excess of the recommended standard deviation by the positive control (19.0%) is still considered acceptable since the positive control produced a distinct positive result with very low absorbance values, and the mean OD values were within the range of historical control data.

Compared to the relative absorbance value of the negative control the mean relative absorbance value was 111.7% after exposure of the skin tissues to the test item and after taking the correction factor into consideration. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.