Aluminium Bordeaux

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2016 until 02 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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)

Remarks:

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

Test material

Test material information

1

Reference substance name:

disodium 2,4-dihydroxy-5-[(2-hydroxy-3-nitro-5-sulphonato-phenyl)azo]benzoate, reaction products with chromium triacetate

Molecular formula:

not applicable

IUPAC Name:

disodium 2,4-dihydroxy-5-[(2-hydroxy-3-nitro-5-sulphonato-phenyl)azo]benzoate, reaction products with chromium triacetate

In vitro test system

Test system:

human skin model

Vehicle:

other: DPBS (MatTek)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg (~ 39 mg/cm²)

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: DPBS (MatTek)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- 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 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 as positive control per tissue.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: absorbance

Run / experiment:

mean of all

Value:

ca. 77

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 (exposure interval of 60 minutes):

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Absor-bance of 3 Wells	Mean Ab-sorbance of 3 wells blank corrected	Mean Absorbance	Rel. Absor-bance [%] Tissue 1, 2 + 3*	Rel. Standard Deviation [%]	Mean Rel. Ab-sorbance [% of Negative Control]**	Mean Absorbance Value Correction Procedure	Mean Rel. Absorbance [% of Negative Control] after Colour Interference and MTT Reduction Correction
Blank		0.037	0.038	0.036	0.037	0.000
Negative Control	1	1.769	1.680	1.727	1.725	1.688	1.609	105.0	5.7	100.0
	2	1.582	1.538	1.517	1.546	1.509		93.8
	3	1.687	1.642	1.667	1.666	1.629		101.2
Positive Control	1	0.095	0.095	0.096	0.095	0.058	0.052	3.6	11.8	3.2
	2	0.087	0.088	0.087	0.087	0.050		3.1
	3	0.083	0.084	0.083	0.083	0.047		2.9
Test Item	1	1.201	1.193	1.197	1.197	1.160	1.244	72.1	5.8	77.3	1.244-0.014– (0.064-0.067) = 1.233	76.7
	2	1.340	1.299	1.329	1.323	1.286		80.0
	3	1.373	1.281	1.310	1.322	1.285		79.9
Blank		0.035	0.035	0.036	0.036	0.000
Add. Viable TissueNeg.Contr.without MTT	1	0.046	0.045	0.045	0.045	0.009	0.009	0.6	0.5	0.6
	2	0.045	0.045	0.045	0.045	0.009		0.6
Add. Viable TissueTest itemwithout MTT	1	0.048	0.051	0.047	0.049	0.013	0.014	0.8	0.9	0.9
	2	0.050	0.052	0.049	0.050	0.015		0.9
Blank		0.035	0.035	0.036	0.036	0.000
Add. Freeze-killed TissueNeg.Contr.	1	0.105	0.104	0.104	0.104	0.069	0.067	4.3	4.4	4.1
	2	0.102	0.099	0.100	0.100	0.065		4.0
Add. Freeze-killed TissueTest item	1	0.103	0.098	0.100	0.100	0.065	0.064	4.0	0.4	4.0
	2	0.105	0.097	0.098	0.100	0.064		4.0

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

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:

Thisin vitrostudy was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

Due to the intensive intrinsic colour of the test item, additional tests with viable tissues (without MTT addition) and with freeze-killed tissues were conducted to determine correction factors for calculating the true viability in the main test.

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 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 41 hours the tissues were treated with the MTT solution for 3 hours following 2.5 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 3.2% 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 12% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative controlthe corrected mean relativeabsorbance value was reduced to 76.7% 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.