Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-10-04 until 2018-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: • MatTek Corporation Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 07 November 2014
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 640/2012, L 193, Part B. 46. “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (06 July 2012).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

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

In vitro test system

Test system:
human skin model
Remarks:
human reconstructed epidermis model
Vehicle:
unchanged (no vehicle)
Details on test system:
Epi-200- SIT Kit (Lot No.: 28665):

1 Sealed 24-well plate, Contains 24 inserts with EpiDerm™ tissues on agarose

2 24-well plates, For MTT viability assay

8 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium, DMEM-based medium
1 bottle DPBS Rinse Solution, for rinsing the inserts in MTT assay
1 vial 5% SDS Solution (TC-SDS-5%), Skin irritant reference chemical –


MTT-100 Assay Kit Components:

1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM), for diluting MTT concentrate prior to use in the MTT assay
1 bottle Extractant Solution (Isopropanol), for extraction of formazan crystals
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Negative Controls: 30 µL were applied to each of triplicate tissues
Positive Controls: 30 µL were applied to each of triplicate tissues
Test Material: Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues
Duration of treatment / exposure:
60 minutes.
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Test animals

Species:
other:

Test system

Type of coverage:
other: topical
Preparation of test site:
other:
Vehicle:
other:

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissues
Value:
71.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
viable tissues
Value:
0.33
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Freeze killed tissues
Value:
2.37
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Non specific killed controls
Value:
0.46
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Other effects / acceptance of results:
Acceptance Criteria:
Criterion 1 (negative control): The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is  0.8 and ≤ 2.8.
Criterion 2 (positive control): An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is  20%.
Criterion 3 (standard deviation): The SD of 3 identical replicates should be ≤ 18.
Criterion 4: OD values should not be below historically established boundaries.
Concurrent negative controls (NC) and positive controls (PC) will be used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues are within a defined historical acceptance range.
Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness are annexed to the report, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiDermTM lot. According to the OECD TG 439, the acceptance limit of the ET50 should be between 4.77 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria).

Any other information on results incl. tables

Results after treatment with Reactive Blue F07 -0195 and the controls

       

Mean 

Mean OD  Mean  Relative   Mean 
  OD of 3 wells OD of Viability Relative
  OD  OD  OD  of blank 3 tissues  [%] Tissue Viability
Treatment Group Tissue No. 570 nm 570 nm 570 nm 3 Wells corrected blank corrected 1, 2 + 3* Standard Deviation [%]**
    Well 1 Well 2 Well 3            
Blank   0.036 0.037 0.038 0.037  
  1 2.028 2.108 2.148 2.095 2.058 105.412  
Negative Control 2 1.906 1.917 1.886 1.903 1.866 1.952 95.587 5.0 100.0
  3 1.985 1.969 1.955 1.970 1.933   99.001    
  1 0.090 0.095 0.095 0.093 0.057 2.894  
Positive Control 2 0.103 0.099 0.097 0.100 0.063 0.060 3.224 0.2 3.08
  3 0.099 0.098 0.098 0.098 0.061   3.131    
  1 1.504 1.611 1.669 1.595 1.558 79.811  
Test Item 2 1.321 1.383 1.378 1.360 1.323 1.381 67.789 8.0 71.30***
  3 1.281 1.298 1.320 1.300 1.263   64.690    
Blank   0.038 0.037 0.037 0.037  
Negative Control 1 0.045 0.045 0.046 0.045 0.008 0.009 0.410 0.1 0.470
Viable Tissues 2 0.049 0.047 0.047 0.048 0.010   0.529    
Test Item  1 0.043 0.043 0.044 0.043 0.006 0.006 0.299 0.0 0.33
Viable Tissues 2 0.044 0.044 0.045 0.044 0.007   0.352    
Negative Control 1 0.091 0.093 0.092 0.092 0.055 0.054 2.804 0.0 2.78
Freeze killed Tissues 2 0.091 0.089 0.093 0.091 0.054   2.747    
Test Item 1 0.085 0.084 0.085 0.085 0.047 0.046 2.421 0.1 2.37
Freeze Killed Tissues 2 0.083 0.082 0.083 0.082 0.045   2.312    
Test Item NSKC 1 0.045 0.046 0.045 0.045 0.008 0.009 0.417 0.1 0.460
2 0.043 0.046 0.052 0.047 0.010   0.497    

*                      Relative viability [rounded values]:            

**                    Mean relative viability [rounded values]:   

***                  corrected value

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water resulted in blue colour due to the intrinsic colour of the test item itself.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did showed blue/purple colour.

The mean relative viability of the test item, corresponding to cell viability, decreased to71.3% (threshold for irritancy:50%), consequently the test item was declared as non-irritant to skin. 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
not irritant to skin according to UN GHS and EU CLP regulation
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, Reactive Blue F07-0195 is not irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of Reactive Blue F07-0195 by means of the Human Skin Model Test.

The test item reduced MTT (test for direct MTT reduction), and its intrinsic colour was intensive (test for colour interference). Consequently, additional tests with freeze-killed and viable tissues were necessary.

Each three tissues of the human skin model EpiDermwere treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item Reactive Blue F07-0195 the mean relative viability value decreased to 71.3% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reactive Blue F07-0195 is non-irritant to skin.