Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Feb - 21 Mar 2016
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)
Version / remarks:
adopted 28 July 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)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, 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™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue lot number: 23324
- Delivery date: 15 March 2016
- Date of initiation of testing: 15 March 2016

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
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- 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.
- MTT concentration:1 mg/mL
- Incubation time: 3 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. The determined OD (540 - 570 nm) was 1.862 ± 0.078 (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 5.6 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 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
- Concentration: 5% aqueous solution
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
Remarks:
mean of 3 tissues
Run / experiment:
60 min exposure
Value:
71.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour. Thus, the test substance was not considered to be a MTT reducer.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test substance's colour change potential in water did not led to a change in colour.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.635, 1.976 and 1.715) was in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min 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 6.2% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variabilities of the test substance, the positive and negative controls were <14%, thus ensuring the validity of the study.

Any other information on results incl. tables

Table 2. Results after 60 min incubation time.

Test group

Mean absorbance at 570 nm

Mean absorbance of three tissues

Rel. absorbance (%) Tissue 1, 2 and 3**

Rel. standard deviation (%)

Mean rel. absorbance (% of negative control)***

Tissue 1*

Tissue 2*

Tissue 3*

Negative control

1.635

1.976

1.715

1.775

92.1
111.3
96.6

10.0

100.0

Positive control

0.124

0.095

0.111

0.110

7.0
5.3
6.3

13.2

6.2

Test substance

1.369

1.170

1.285

1.275

77.1
65.9
72.4

7.8

71.8

* Mean of 3 replicate wells after blank correction

** 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:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the Reconstructed Human Epidermis test the test substance does not possess any skin irritating potential.