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Description of key information

Skin Irritation

In Vitro test Skin irritation was choosen as a key study. The test was performed according totheOECDTest GuidelineNo.439:In VitroSkin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

GLP study.

Klimish score 1.

Eye Irritation

In vivo test Eye Irritation was choosen as a key study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28.03.-02.06.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 30.6.2016TEMPERATURE 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
Irritation / corrosion parameter:
% tissue viability
Value:
101.1
Negative controls validity:
valid
Positive controls validity:
valid
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.

Table No. 1: OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities 

 

Treatment

OD570

Avg

SD

Average viability

Code

1

2

3

(% NC)

NC

 

PBS

1.961

1.946

1.941

1.949

0.008

100.0 

% of avg NC

100.6

99.8

99.6

100.0

0.4

C4

 

595/16

2.093

2.047

1.775

1.972

0.140

101.1 

% of avg NC

107.4

105.0

91.1

101.1

7.2

C4

CC

 

595/16

0.010

0.004

 -

0.007

0.003

0.4 

% of avg NC

0.5

0.2

-

0.4

0.2

PC

 

5% SDS

0.059

0.053

0.056

0.056

0.002

2.9

% of avg NC

3.0

2.7

2.9

2.6

0.1

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Direct Blue 78 was 101.1 % of negative control average value i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model. According to the classification criteria given in chapter 4.5 of this report, the test substance, Direct Blue 78, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test substance, Direct Blue 78, 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 EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

Colour interference was performed simultaneously. The test substance did not interfere with the endpoint.

Direct MTT reduction in the test tube was not performed because of blue colour of solutions of the test substance. Direct MTT test in frozen tissues excluded direct MTT reduction caused by the test substance. 

After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissuesurface. 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 designaverage viability of treated tissues was101.1%,i.e. viability was >50 %.

The effect of the test substance was negative in EpiDermTMmodel (tissues were not damaged).

According to the classification criteria given in chapter 4.5. of this report,the test substance, Direct Blue 78, is considered to have no category in regard to skin irritation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.-03.1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards and acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The study report contains the limited information on the test conditions because the study has been performed in 1991 . However available information is sufficient to conclude on the classification of the substance. Further the test was performed on the vertebrates and use of results from old experimental studies is one of the options to provide information requested by REACH. New experimental studies with vertebrates must only be conducted if there is no adequate existing information.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Controls:
not specified
Irritation parameter:
other: cornea damage
Basis:
mean
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
other: mucosa demage
Basis:
mean
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
The substance is nonirritating.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

Based on available data the test substance, Direct Blue 78, is not classified as a skin irritant.

Based on available data the test substance, Direct Blue 78, is not classified as a eye irritant.