Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 April 2016 until 06 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

EC Number:

276-817-3

EC Name:

Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

Cas Number:

72749-80-5

Molecular formula:

C28H20N4O6S2.2Na

IUPAC Name:

Benzo[a]phenazinium, 9-amino-7-phenyl-5-(phenylamino)-4,10-disulfo-, inner salt, sodium salt (1:2)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Purity: 94.7% (w/w), dose calculation was not adjusted to purity
Expiry Date: 09 March 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 triplicate tissues wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

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:

- Amount(s) applied (volume or weight with unit):
-Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL of DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL DPBS (MatTek) were used as negative control per tissue.

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL of a 5% SLS solution in deionised 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:

The test item, the negative control (DPBS) and 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 approximately 69 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Relative Absorbance (%)

Run / experiment:

1

Value:

105.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

other: Relative Absorbance (%)

Run / experiment:

2

Value:

112.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

other: Relative Absorbance (%)

Run / experiment:

3

Value:

112.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results after treatment with the test item and the controls:

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Ab-sor-bance of 3 Wells	Mean Ab-sorbance of three wells blank corrected	Mean Absorbance of 3 wells after blank correction*	Rel. Absor-bance [%] Tissue 1, 2 + 3*	Rel. Stand. De-viation [%]	Mean Rel. Absorbance [% of Negative Control]**	Mean Absorbance Blank Corrected Viable Tissue without MTT (Step 2)	Mean Absorbance Blank Corrected Freeze-killed Tissue With MTT (Step 4)	Mean Rel. Absorbance [% of Negative Control] after Complete Correction Procedure
Blank contr.		0.036	0.037	0.036	0.036	0.000
Blank TI		0.037	0.038	0.038	0.037	0.000
Negative Control	1	1.578	1.549	1.558	1.562	1.525	1.811	84.2	13.7	100.0
	2	2.026	1.984	1.971	1.994	1.957		108.1
	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	19.0	4.3
	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
Test Item	1	2.012	1.921	1.929	1.954	1.917	1.996	105.8	3.4	110.2	0.149	0.348	85.8
	2	2.082	2.087	2.051	2.073	2.036		112.4
	3	2.093	2.067	2.059	2.073	2.036		112.4

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

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, Duasyn Acid Violet SD 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.

Since the test item was deeply coloured (black), a change of colour in the MTT interference pre-test to detect the test item’s property directly reducing MTT was not visible, if there was any. Therefore, as a precaution, the additional test with freeze-killed tissues was performed for possible data correction in the main experiment. Due to its intensive colour and for the same purpose, also the additional test with one viable tissue but without MTT addition had to be performed (test for colour interference).

Each three tissues of the human skin model EpiDerm^™were treated with the test item, the negative or the positive control for 60 minutes.

Approximately 25 mg of the test item were applied to each tissue and spread to match the surface of triplicate tissue.

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

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

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

Taking the results in the additional tests with viable and freeze-killed tissues into consideration, the mean relative absorbance of the test item amounted to 85.8% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.