Reaction products of 2-hydroxy-6-nitro-4-sulphonaphthalene-1-diazonium coupled with resorcinol, subsequently coupled with diazotised 5-amino-2-anilinobenzenesulphonic acid, iron(III) complexes, sodium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14th August 2018 to 14th September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 439

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

Version 439, adopted 28. July 2015

Deviations:

yes

Remarks:

Not critical.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III.
Adopted 06. Jul. 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

pentasodium bis 4-((2,4-dihydroxy-5-((4-(phenylamino)-3-sulfonatophenyl)diazenyl)phenyl)diazenyl)-3-hydroxy-7-nitronaphthalene-1-sulfonate ferrate

EC Number:

947-799-0

Molecular formula:

C56H32FeN12O22S4.5Na

IUPAC Name:

pentasodium bis 4-((2,4-dihydroxy-5-((4-(phenylamino)-3-sulfonatophenyl)diazenyl)phenyl)diazenyl)-3-hydroxy-7-nitronaphthalene-1-sulfonate ferrate

Details on test material:

The test material is a UVCB substance.

Specific details on test material used for the study:

TEST ITEM
Name: ACID BROWN 396:1
Batch no.: No. 77171
Appearance: Brown powder
Purity: 100% (UVCB)
Homogeneity: homogeneous
EINECS-No.: 947-779-0

STABILITY AND STORAGE OF THE TEST SYSTEM
Storage Room: Temperature (20 ± 5 °C)

In vitro test system

Test system:

human skin model

Remarks:

EpiDermTM-Kit, procured by MatTek

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

other: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium)

Remarks:

Procured by MatTek In Vitro Life Science Laboratories, batch no.: 090618MSA

Details on test system:

TEST SYSTEM SPECIFICATION
The test system is a commercially available EpiDermTM-Kit (EPI-200-SIT), procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.

TEST VESSELS
All vessels used are made of glass or sterilised plastic. The glassware was sterilised before use by autoclaving. The following vessels were used: 6-well-plates, 24-well-plates, 96-well-plastes.

PRE-INCUBATION OF TISSUES
All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator for 1 hour. After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium. Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator for 1 hour.

TREATMENT
One plate (3 tissues) was used as negative control; each tissue was treated with DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 μL 5% SDS-soluttion, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used for treatment with the test item: The tissues were wetted with DPBS buffer before applying the test item and spreading it to match the tissue size.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The following amounts were applied to the tissues:
Tissue Amount
1 26.2 mg
2 25.3 mg
3 25.8 mg

Duration of treatment / exposure:

Tissues were dosed in 1-minute intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at controlled conditions. 1 hour after the first application, the inserts were removed from the plates and rinsed immediately in 1-minute intervals.The surface of the inserts was then carefully dried. Then, the tissues were set in the incubator for further 23 hours.

Duration of post-treatment incubation (if applicable):

Post-incubation lasted 17 hours and 35 minutes into the incubator.

Number of replicates:

2 measurements per 3 tissues.
As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 98

Remarks on result:

no indication of irritation

Any other information on results incl. tables

EVALUATION

The values of the 96-well-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).

COMPARISON OF TISSUE VIABILITY

For each replicate of test item and positive control, tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls.

The following percentage values of tissue viability were calculated in comparison to the negative control:

Designation	ACID BROWN 396:1	Positive Control
% Tissue viability (tissue 1)	93.7%	4.1%
% Tissue viability (tissue 2)	100.6%	3.7%
% Tissue viability (tissue 3)	99.6%	3.3%
% Tissue viability (mean)	98.0%	3.7%
± SD of mean tissue viability (%)	3.7%	0.4%

 

VALIDITY OF THE TEST

Validity criteria and results are stated in the following table:

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.8	1.6
% tissue viability of positive control SDS	≤ 20% of negative control	3.7%
SD of mean viability of the tissue replicates (%)	≤ 18%	5.5% (negative control) 0.4% (positive control) 3.7% (test item)

All validity criteria were met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Non-irritant to skin

Conclusions:

The test item ACID BROWN 396:1 is considered as non-irritant to skin.
After the treatment, the mean value of relative tissue viability was reduced to 98.0%. This value is above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.
Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid.

Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDermTM were treated with the test item for 1 hour. The test item was applied directly to each tissue and spread to match the tissue size. DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.6. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 3.7% (required ≤ 20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 98.0%. This value is above the threshold for skin irritation potential (50%).

Therefore, the test item ACID BROWN 396:1 is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method because is tissue viability is > 50%.