Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

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)

Rationale for reliability incl. deficiencies:

other: 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. 2009)

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

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

287-267-9

EC Name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzylidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

85455-32-9

Molecular formula:

C13H7CrN3O10S.C11H25NO.H

IUPAC Name:

Hydrogen hydroxy[2-hydroxy-3-[(2-hydroxy-3-nitrobenzy-lidene)amino]-5-nitrobenzenesulphonato(3-)]chromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Test material form:

solid: particulate/powder

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 of DPBS, 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

Details on study design:

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative and the positive control for 60 minutes.
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 tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue 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 69.75 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

Irritation / corrosion parameter:

% tissue viability

Remarks:

expressed as mean relative absorbance

Run / experiment:

all 3

Value:

> 97.8 - < 105.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Results after treatment with the test item and the controls

 

Dose Group	Ex-po-sure Interval	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 Wells	Mean-Absorbance of three wells blank corrected	Mean Absorbance of 3 wells after blank correction*	Rel. Absorbance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]**	Mean Absorbance Blank corrected viable tissue without MTT (Step 2)	Mean Rel. Absorbance [% of Negative Control] after colour interference and MTT reduction correction
Blank			0.036	0.037	0.036	0.036	0.000
Negative Control	60 min	Tissue 1	1.736	1.703	1.695	1.711	1.675	1.673	100.1	1.1	100.0
		Tissue 2	1.679	1.688	1.699	1.689	1.653		98.8
		Tissue 3	1.725	1.713	1.740	1.726	1.690		101.0
Positive Control	60 min	Tissue 1	0.116	0.116	0.118	0.117	0.081	0.075	4.8	6.6	4.5
		Tissue 2	0.107	0.111	0.109	0.109	0.073		4.4
		Tissue 3	0.108	0.105	0.110	0.107	0.071		4.3
Test Item	60 min	Tissue 1	1.800	1.774	1.835	1.8037	1.767	1.711	105.7	4.0	102.3	0.009	101.8
		Tissue 2	1.677	1.685	1.654	1.672	1.636		97.8
		Tissue 3	1.765	1.758	1.775	1.766	1.730		103.4

 

* relative absorbance per tissue [rounded values]: 100 x (absorbance_tissue) divided by mean absorbance _{negative control}

** relative absorbance per treatment group [rounded values]: 100 x (mean absorbance _{test item/positive control}) divided by (mean absorbance _{negative control})

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, 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 interference pre-test. Due to its intensive colour, an additional test with one viable tissue (without MTT addition) was necessary to correct the result 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 tissue.

30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to triplicate tissue 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 42 hours the tissues were treated with the MTT solution for 3 hours following 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 4.5% 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 below 7% (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 mean relative absorbance value was not reduced (101.8%) 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.