Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 - 22 Sep 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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 26 July 2013
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)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

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:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 19674 Kit B
- Delivery date: 17 Sep 2014
- Date of initiation of testing: 17 Sep 2014

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 hood

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 was prepared on the day of testing using MTT-100 Assay Kit Components.
- Incubation time: 42 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 ± 1 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.
- 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 6.5 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 directly reduce MTT, 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
- The test substance is considered to be irritant to skin 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
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
approximately 42 h
Number of replicates:
triplicates for each treatment and control group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
101
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The test substance was not considered to be a MTT reducer.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.742, 1.808 and 1.904) was in the range of ≥ 0.8 and ≤ 2.8 for 60 minutes treatment, thus showing the quality of 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 6.3% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The standard deviations of the % variabilities of the test substance, the positive and negative controls were < 10%, thus ensuring the validity of the study.

Any other information on results incl. tables

Table 2. Results after treatment

 Dose Group

 Treatment Interval

Absorbance 570 nm Tissue 1*

Absorbance 570 nm Tissue 2*

Absorbance 570 nm Tissue 3*

Mean Absorbance of 3 Tissues 

Rel. Absorbance [%] Tissue 1, 2 + 3** 

Relative Standard Deviation [%] 

Mean Rel. Absorbance [% of Negative Control]*** 

Negative Control

 60 min

 1.742

 1.808

 1.904

 1.818

95.8; 99.5; 104.7

 4.5

 100.0

Positive Control

 60 min

 0.126

 0.117

 0.103

 0.115

6.9; 6.4; 5.7

 9.7

 6.3

Test Item

 60 min

 1.889

 1.740

 1.877

 1.835

103.9; 95.7; 103.2

4.5 

101.0 

* Mean of three replicate wells after blank correction

** relative absorbance per tissue [rounded values]: (100 x (absorbance tissue)) / (mean absorbance negative control))

*** relative absorbance per treatment group [rounded values]: (100 x mean absorbance test item/positive control) / (mean absorbance negative control)

 

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. The mean relative absorbance value of the test item, corresponding to the cell viability, was not reduced (101.0%; threshold for irritancy: ≤ 50%), consequently, the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
In the skin irritation study in vitro, the treatment with the test substance resulted in a cell viability of 101% and thus the test substance does not possess any skin irritating potential.