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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 November 2012 to 30 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The reliability is rated 1 because the study followed the standard guideline of reference (OECD 439), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD guideline 439 (In-vitro skin irritation)
Qualifier:
according to guideline
Guideline:
other: EU method B.46 (In vitro skin irritation)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
anilino(phenylimino)methanaminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
EC Number:
941-151-0
Molecular formula:
C31H28N6SO3
IUPAC Name:
anilino(phenylimino)methanaminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):Sepisol Fast Yellow MG-DPG
- Substance type: Organic salt
- Physical state: Powder
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Test animals

Species:
human
Strain:
other: reconstructed epidermis of normal human keratinocytes.
Details on test animals or test system and environmental conditions:
TEST SYSTEM
- Source: episkin snc - 4 rue Alexander Fleming - 69366 Lyon CEDEX 07.
- Cell system used: reconstructed epidermis of normal human keratinocytes. Cells are grown on inert polycarbonate filter on chemically defined medium, arilifted for 17 days.
- RhE model : SkinEthic RHE/S/17
- Lot/Batch No.: 12 022A 1103


Test system

Type of coverage:
open
Preparation of test site:
other:
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Amount / concentration applied:
16 mg applied as such to the epidermal surface
Duration of treatment / exposure:
not applicable
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
I- TEST SYSTEM

Units of 0.50 cm² reconstituted epidermis (SkinEthic) were received on the day before the experiment. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter.

II- CHECK OF THE COMPATIBILITY OF THE TEST ITEM (direct MTT reduction, coloring potential, ability to stain tissues) WITH MTT SOLUTION

Prior to experiment, 16 mg of the test item was put in contact with 300 µL of MTT solution (1 mg/mL). After a 3 hrs incubation period at 37°C, 5% CO2, no blue or purple coloration was noted.

Therefore, there is no direct interaction between the test item and MTT.

III- APPLICATION OF THE TEST AND CONTROL CHEMICALS

* For all steps of the protocol, one 24 wells plate (3 columns of 4 wells) was used for each item (the test item, the negative control and the positive control).

* The insert were placed in a 24 wells culture plated which has been previously filled with 300 µL of maintenance medium (Batch No. 12 011J-M 037).

* 3 units of reconstructed human epidermis were used for the test item, the negative and positive controls.

* For the test item: 16 mg was applied as such to the epidermal surface of the models. For the positive control: XXX µL of 5% SDS (CAS N° 151-21-3, Sigma Batch N° BCBF8969V) were applied to uniformly cover the epidermis surface. For the negative control: XXXX µL of PBS (Pan Biotech GmbH – Batch N° 2020312) were applied to uniformly cover the epidermis surface.

* At the end of an exposure period of 42 min at room temperature, the human epidermis was washed with25 x 1 mL of PSB (PAN BIOTECH GmbH, Batch n° 2020312) except for the three epidermises treated with the test item wich were rinces with 35 x 1 mL of PBS. Even after this rinse, there was remaining test item on the surface of the three epidermises.

* Rinsed epidermis were incubated in fresh medium for a 42 hours 10 min post-treatment incubation period at 37°C, 5% CO2.

* Then, the epidermis were put in contact with the MTT solution.

IV-CELL VIABILITY MEASUREMENTS

* After the 42h10min incubation period, the cell viability was quantified by measurement of the cell succinate dehydrogenase activity. The skin sample was placed in 300 µL of a MTT solution of 1 mg/mL concentration for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product is then extracted using isopropanol during 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extractions in isopropanol (1:2).

The absorbance was measured in triplicate of MTT extract.

The optical density was performed usgin the ELx800 absorbance microplate reader and the validated software Gen5ELISA V1.05.11

V- INTERPRETATION OF RESULTS

* The results were expressed as a viability percentage compared with the negative control :

Viability %= [OD (test item)/OD (negative control)] ×100

The optical density (OD) values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: cell viability percentage
Value:
ca. 86.3
Remarks on result:
other:
Remarks:
Basis: mean. (migrated information)

Any other information on results incl. tables

 

Skin

OD

Mean OD/disc

(Mean of the 3 OD measurements)

Mean OD/ product

Viability %

Mean Viability %

SD

Conclusion

Negative Control

1

1.041

1.100

1.138

1.093

1.031*

106.0

100.0

5.9

 

2

0.961

1.039

1.092

1.030

99.9

3

0.908

0.976

1.029

0.971

94.1

Positive Control

4

0.012

0.014

0.013

0.013

0.013

1.3

1.2

0.1

Irritant

5

0.011

0.013

0.012

0.012

1.2

6

0.013

0.012

0.014

0.013

1.3

Test Item

7

0.216

0.243

0.250

0.236 – Aberrant values

 

 

 

 

 

8

0.763

0.761

0826

0.783

0.890

76.0

86.3*

14.6*

Non irritant

9

0.918

1.030

1.040

0.996

96.6

* The result is based on two tissues replicates (n° 8 and 9) instead of three as initially scheduled in the study plan. The rinsing of the epidermises in view to eliminate the test item after treatment was difficult and required more rinsing with PBS than usually. The first treated epidermis was compromised during this rinsing step and was not taken into account for the classification of the test item.

                 

Negative control and positive control are in the expected range

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results obtained under these experimental conditions, enable to conclude that the test item must not be classified, according to the criteria for classification, packaging and labelling of dangerous substances and preparations in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. No symbol or risk phrase is required.
In accordance with the Regulation EC No. 1272/2008, the test item must not be classified. No signal word or hazard statement is required.
Executive summary:

The aim of the study was to evaluate the possible irritating effects of SEPISOL FAST YELLOW MG-DPG after topical administration on in vitro human reconstructed epidermis (RHE® model). The test item SEPISOL FAST YELLOW MG-DPG was applied, as supplied, at the dose of 16 mg, to 3 Reconstructed Human epidermis small during 42 minutes, followed by a 42 hours postincubation period at 37°C, 5% C02.

The elimination of the test item on the epidermis after treatment was difficult. Even after a rinse with 35 mL of PBS, there was remaining test item. The first epidermis was compromised during rinsing and was not taken into account for the classification of the test item.

The experimental protocol was established in accordance with OECD guideline No. 439 adopted 22 July 2010 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220)-ATP Council regulation No. 4401/2008 of 30 May 2008 (E. U. Journal L142).

The mean viability ofthe epidermis skins was 86.3%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate ).

The results obtained, under these experimental conditions, enable to conclude that the test item SEPISOL FAST YELLOW MG-DPG must not be classified, according to the criteria for classification, packaging and labelling of dangerous substances and preparations in compliance with the E.E.C. Directives 67/548, 2001/59 and 99/45. No symbol or risk phrase is required.

In accordance with the Regulation EC No. 1272/2008, the test item must not be classified. No signal word or hazard statement is required.