Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 April 2016 until 18 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 439 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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)

Deviations:

no

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)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

EC Number:

276-538-7

EC Name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Cas Number:

72252-58-5

Molecular formula:

C23H8Cr2N6O16S3.4Na

IUPAC Name:

tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

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, wetted with 25 µL DPBS prior to application, and spread to match the surface of the tissue

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

Amount / concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS prior to application, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks:

Episkin model, 3 tissues per concentration

Run / experiment:

mean

Value:

90.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Results after treatment with the test item and the controls after 60 minutes exposure interval

Dose Group	Tissue No.	well 1	well 2	well 3	Mean	Mean-Blank*	Mean of three tissues	Stand. Dev.	Rel. Absor-bance [%] Tissue 1, 2 + 3**	Stand. Dev. [%]	Mean Rel. Ab-sorbance [% of Negative Control] ***	Dif-ference 1****	Dif-ference 2*****	Dif-ference 3 ******	Mean OD blank corrected viable tissues without MTT	Mean OD of two killed tissues blank cor-rected treated with the test item	Mean OD of two killed tissues blank corrected treated with negative control	Cor-rected Via-bility Mean Abs. 1 [%]	Corrected Via-bility Mean Abs. 2 [%]	Corrected Via-bility Mean Abs. 3 [%]
Blank		0.037	0.038	0.038	0.038	0.000
Nega-tive Control	1	1.892	1.879	1.888	1.886	1.848	2.019	0.181	91.5	9.0	100.0
	2	2.037	2.064	2.013	2.038	2.000			99.1
	3	2.244	2.243	2.252	2.246	2.209			109.4
Test Item	1	1.740	1.748	1.771	1.753	1.715	1.852	0.121	84.9	6.6	91.8	1.848	1.836	1.832	0.004	0.155	0.139	91.553	90.935	90.737
	2	1.867	1.971	1.966	1.935	1.897			94.0
	3	1.957	1.990	2.003	1.983	1.945			96.4
Posi-tive Control	1	0.104	0.112	0.107	0.108	0.070	0.074	0.004	3.5	5.4	3.7
	2	0.109	0.125	0.112	0.115	0.078			3.8
	3	0.113	0.114	0.113	0.113	0.076			3.8

 

*             Mean of three replicate wells after blank correction

**            relative absorbance per tissue [rounded values]:

***           relative absorbance per treatment group [rounded values]:

 

****        Mean OD viable Tissue (blank corrected from three tissues with three replicates) minus mean OD (three replicates one viable tissue without MTT after blank correction)

*****       Mean OD viable Tissue (blank corrected from three tissues with three replicates) minus (mean OD of two killed tissues treated with test item minus mean OD of two killed tissues treated with negative control)

******      Mean OD viable Tissue (blank corrected from three tissues with three replicates) minus mean OD (three replicates one viable tissue without MTT after blank correction) minus (mean OD of two killed tissues treated with test item minus mean OD of two killed tissues treated with negative control)

Applicant's summary and conclusion

Interpretation of results:

other:

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.

An additional test with one viable tissue had to be performed, due to the intensive black colour of the test item. Since the test item dyed the MTT-solution black in the pre-test for direct MTT reduction also due to its intensive black colour, a possible colour turn into blue or purple as a sign being a MTT reducer was not possible to observe. Therefore, as a precaution, an additional test with freeze-killed tissues was performed.

The test item, the negative control (DPBS) and the positive control (5% SLS) were applied to each of triplicate tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following approximately 68.5 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 3.7% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were 9% at the highest (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value was reduced to 90.7% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.