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Toxicological information

Skin irritation / corrosion

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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:
17.01. - 31.03.2017
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
yes
Remarks:
See any other information...
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: model EpiDermTM- Tissue: The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 25802 kit C)- Date of initiation of testing: 30.6.2016The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37±1°C
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied: 25 gNEGATIVE CONTROLPBS MatTek 101816ZSA, exp. 18/10/2017 - Amount(s) applied: 25 µLPOSITIVE CONTROL5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 031617MGKA, exp. 16/03/2018
Duration of treatment / exposure:
60 min.
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
in the first experiment
Value:
36.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
in the second experiment, after correction
Value:
25.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Direct MTT reduction - functional check in tubes The test was not performed due to blue colour of the test substance. The next step was performed directly.

Any other information on results incl. tables

Table 1: The first experiment: OD570 values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

 

Treatment

OD570

 

 

 

 

Avg

SD

%NC

PBS (NC)

2.022

1.940

2.050

NT

NT

2.004

0.047

 

viability (%NC)

100.9

96.8

102.3

-

-

100.0

2.3

100.0 

394/16 (C4)

0.455

1.727

0.730

0.364

0.416

0.738

0.510

 

viability (%NC)

22.7

86.2

36.4

18.2

20.8

36.9

25.5

36.9 

5% SDS (PC)

0.044

0.051

0.050

NT

NT

0.048

0.003

 

viability (%NC)

2.2

2.5

2.5

-

-

2.4

0.2

2.4

Table 2: The second experiment: OD570 values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

Treatment

OD570

 

 

Avg

SD

%NC

PBS (NC)

1.961

1.946

1.941

1.949

0.008

 

viability (%NC)

100.6

99.8

99.6

100.0

0.4

100.0

394/16 (C3)

0.282*

0.264

1.098

0.548

0.389

 

viability (%NC)

14.5

13.5

56.3

28.1

20.0

28.1

394/16 (C3_CC)

0.049*

0.042+

NT

0.046

0.004

 

viability (%NC)

2.5

2.2

 -

2.3

0.2

2.3

5% SDS (PC)

0.059

0.053

0.056

0.056

0.002

 

viability (%NC)

3.0

2.7

2.9

2.9

0.1

2.9

NC, PC

negative, positive control

C3, C4, 394/16

test substance 

avg

arithmetic average

SD

standard deviation calculated from individual % tissue viabilities

viability (%)

viability of single tissues compared with negative control

NT

not tested

*

damaged tissue (with lost of part of tissue)

+

damaged tissue (without lost of part of tissue)

Applicant's summary and conclusion

Interpretation of results:
other: The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Acid Black 227 was 36.9 % and 25.8% of negative control average value i.e. viability was < 50 % in both cases. The effect of the test substance was positive in EpiDermTM model. The test substance, Acid Black 227, is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.
Executive summary:

The test substance, Acid Black 227, was assayed for in vitro skin irritation in the human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

In the preliminary experiments neither colour interference with the endpoint nor direct MTT reduction were found.

After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design average viability of treated tissues was 36.9 % in the first and 25.8 % (after correction) in the second experiment, i.e. viability was < 50 %.

The effect of the test substance was positive in EpiDermTM model (tissues were damaged).

The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.