Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Test material form:
solid

In vitro test system

Test system:
human skin model
Vehicle:
unchanged (no vehicle)
Details on test system:
Three dimensional human epidermis model
The EpiDermTM model consists of normal, 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 arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: EPI-200
Tissue Lot Number: 23377 (test run 1) and 23385 (test run 2) (Certificates of Analysis see appendix)
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively.
In addition three killed control tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction.
25 µL sterile PBS was applied first. As the test substance could not be applied with a sharp spoon, a metal pin was covered with a bulk volume of ca. 25 µL of the solid test material and was applied with direct contact to the tissue.
Control tissues were concurrently treated with 30 µL of sterile PBS (NC, NC KC) or with 30 µL of 5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue surface of the NC, NC KC and PC afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.


Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 25µl bulk volume (about 44 mg).


Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
92

Any other information on results incl. tables

Irritation test: 2nd test run

Test substance identification

 

 

tissue 1

tissue 2

tissue 3

mean

SD

CV[%]

NC

viable tissues

mean OD570

 

1.980

1.997

1.873

 

 

 

viability

[% of NC]

101.5

102.4

96.1

100.0

3.4

3.4

KC tissues

mean OD570

 

0.045

0.049

0.048

0.047

 

 

viability

[% of NC]

2.3

2.5

2.5

2.4

0.1

5.0

16/0255-1

viable tissues

mean OD570

 

1.742

1.760

1.888

1.797

 

 

viability

[% of NC]

89.3

90.2

96.8

92.1

4.4

4.4

KC tissues

mean OD570

 

0.000

0.000

0.006

0.002

 

 

viability

[% of NC]

0.0

0.0

0.3

0.1

0.2

173.2

Final mean viability of tissues after KC correction[% of NC]:

92.0

 

 

PC

viable tissues

mean OD570

 

0.047

0.046

0.052

0.049

 

 

viability

[% of NC]

2.4

2.4

2.7

2.5

0.2

6.6

* Negative values are set to zero for further calculation

The tissues were black discolored and compound residues remained on the tissues after the washing procedure.

Due to the intense color of the test substance, the ability of the test substance to reduce MTT directly could not be determined in a pretest. Therefore KC tissues were applied in parallel.

The results of the KC tissues indicate an increased MTT reduction (mean viability 0.1% of NC). Thus for the test substance the final mean viability is given after KC correction.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria, it was concluded, that Direct Black 18L NA active dye does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.