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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:
2016-04-28 to 2016-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Epi-200 kits purchased from MatTek Corporation (Bratislava, Slovakia), containing EpiDerm™ tissues on agarose.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues (Epi-200 SIT kits, MatTek)
- Tissue batch number(s): Lot No.: 23339
- Delivery date: 19 January, 2016
- Date of initiation of testing: 19 January, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, remaining period of treatment at room temperature on a sterile bench
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: rinsing with DPBS at least 15 times, followed by submerging of inserts in DPBS at least 3 times, then additional rinsing with sterile DPBS (volume not stated)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: Versamax®Molecular Devices, Softmax Pro (version 4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Positive control: mean viability: 4.77%; relative standard deviation: 14.9%; range of viabilities: 4.00-5.90%.
-Absorption:
Positive control: mean absorption: 0.084; relative standard deviation: 16.6%; range of absorbance: 0.069-0.097
Negative control: mean absorption: 1.77; relative standard deviation: 8.55%, range of absorbance: 1.66-1.98

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Yes. Evaluation of colour interference: 30 µL of the test item mixed with 300 µL deionised water was incubated for 60 minutes at 37 ± 1.5 °C. Since the colour of the test item mixture did not change during the incubation period, the pre-experiment for colour interference was not continued.
Evaluation of direct reduction of MTT: 30 µL of the test item added to MTT (1 mg/ml). Incubated in the dark at 37 ± 1.5 °C for 60 minutes. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer.

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item is determined and classification is recommended if the mean relative tissue viability of three individual tissues is reduced to less than or equal to 50% of the negative control. The following in vitro results have been related to the following in vivo predictions:
Mean tissue viability =50% = irritant (I)
Mean tissue viability >50% = non-irritant (NI)

Acceptability of the Assay
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 at or below 20%.
Criterion 3 (standard deviation): The SD of 3 identical replicates should be < 18%.
OD values should not be below historically established boundaries.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL (47 µL/cm2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (MatTek) was used as a negative control per tissue.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% SLS solution in deionised water (MatTek) was used as a positive control per tissue.
Duration of treatment / exposure:
60 minutes.
Duration of post-treatment incubation (if applicable):
41 hours and 37 minutes
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
4.3
Other effects / acceptance of results:
- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT: test item did not change colour in the colour interference test
- After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD = 0.8 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, and thus assuring the validity of the test system; the mean relative absorbance value of the positive control was 4.3% compared to the negative control.

In vivo

Irritant / corrosive response data:
The test item is considered to possess an irritant potential.

Any other information on results incl. tables

Results after treatment with test substance and the controls (exposure interval of 60 minutes):

 Dose Group  Tissue No.:

Absorbance

570 nm

Well 1

 Absorbance

570 nm

Well 2

 Absorbance

570 nm

Well 3

 Mean

Absorbance

of 3 Wells

 Mean Absorbance

of 3 wells

blank corrected

 Mean Absorbance

of 3 Tissues

after blank correction

Relative

Absorbance [%]

Tissue 1,2,3 

Relative Standard

Deviation [%] 

 Mean Relative

Absorbance

[% of Negative Control]

 Blank   0.036 

 0.037

0.036   0.036  0.000        
 Negative Control  1  1.960  1.998 1.990  1.982  1.946     102.9    
   2 1.912  1.866  1.865  1.881   1.845 1.891   97.6 2.7  100.0 
   3 1.935   1.916  1.903 1.918   1.881   99.5  

 

 Positive Control

 1

 0.113

 0.113

0.119 

 0.115

0.079 

 

4.2 

 

 

 

 2

0.120 

 0.119

0.122 

0.120 

0.084 

0.081 

4.4 

3.3 

4.3 

 

 3

0.117 

 0.116

0.116 

 0.116

0.080 

 

4.2

 

 

 Test Item

 1

0.108 

 0.115

0.117 

 0.113

0.077 

 

4.1

 

 

 

 2

0.112 

 0.112

0.118 

 0.114

0.078 

 0.081

4.1 

 8.6

4.3 

 

 3

 0.124

 0.126

 0.128

0.126 

0.089 

 

4.7 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
It can be stated that in this study and under the experimental conditions reported, the test substance is an irritant to skin according to UN GHS and EU CLP regulation. However, on their own these data are not sufficient to resolve between category 1 and 2.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item passed the MTT- and the Colour Interference pre-tests. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.8 and = 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.3% thus ensuring the validity of the test system.

The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 9% (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative controlthe mean relative absorbance value was reduced to 4.3% after exposure of the skin tissues to the test item. This value is below the threshold for irritancy of = 50%. Therefore, the test item is considered to possess an irritant potential.