Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Oct - 04 Nov 2016
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 Jul 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008, 1st ATP 2009
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: EpiDerm™ (EPI-200)
Source strain:
other: human
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Human
- Tissue: normal epidermal keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23372
- Delivery date: 01 Nov 2016
- Date of initiation of testing: 01 Nov 2016

EPIDERM™ QUALITY CRITERIA
- Air bubbles between agarose and insert were not >30% of the total surface
- Liquid on top of the insert was removed with sterile cotton tips
- If moisture was observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench
- Temperature of post-treatment incubation: 37 ± 1.5 °C for 42 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.587 ± 0.089 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.25 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not show color interference 1h after incubation in deionised water, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin (Cat 2) if the viability after 1 hour exposure is less than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µl

NEGATIVE CONTROL
- Amount applied: 30 µl

POSITIVE CONTROL
- Amount applied: 30 µl
- Concentration: 5% aequeous solution
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
about 42 h
Number of replicates:
triplicates for each treatment and control group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
60 min exposure
Value:
88.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The test substance was not considered to be a MTT reducer.
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also it intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control ODs (values between 1.910 and 2.227) were in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.1% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations of the 3 identical replicates of the test substance and negative control in the main test were below 7% (threshold of OECD 439: <18%), thus ensuring the validity of the study.

Any other information on results incl. tables

Table 2. Results after treatment with the test substance and controls

 

 

Mean absorbance at 570 nm *

 

 

Mean absorbance

of 3 tissues blank corrected

Rel. absorbance (%) **

Rel. SD (%)

Rel. absorbance (% of negative control)***

Tissue 1

Tissue 2

Tissue 3

Tissue 1

Tissue 2

Tissue 3

Negative control

1.978

2.115

1.902

1.998

99.0

105.8

95.2

5.4

100.0

Positive control

0.064

0.062

0.057

0.061

3.2

3.1

2.9

5.8

3.1

Test substance

1.899

1.765

1.653

1.772

95.0

88.3

82.7

6.9

88.7

* Mean of 3 replicate wells after blank correction (mean blank value: 0.038)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test item/positive control) / (mean absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the conducted test, the test substance did not possess irritating properties towards reconstructed human epidermis tissue in the EpiDerm™ model.
Executive summary:

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

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. Each 30 µL of the test item, of the negative control (DPBS), or of the positive control (5% SLS) were applied to each of triplicate tissue. After treatment with the negative control the absorbance values were well within 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 (3.1%) as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 88.7% 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.