Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Jan - 18 Feb 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 27 July 2015
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™
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: 23314
- Delivery date: 16 February 2016
- Date of initiation of testing: 16 February 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 minutes exposure), 37 ± 1.5 °C (60 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed using a wash bottle containing DPBS to remove any residual test material (20 times).

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 Enterprise v.4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

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 6.55 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: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 2
- Method of calculation used: Since the MTT reducing test substance extract was classified as corrosive by the skin irritation test (tissue viability <50 %), the correction procedures were not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment with 2 incubation periods

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N
Duration of treatment / exposure:
3 and 60 min
Number of replicates:
in duplicates for each treatment and control group

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
3 minutes exposure
Value:
39.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
60 minutes exposure
Value:
10.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
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 showed blue colour. An additional test with freeze-killed tissues had to be performed, but correction of the viability values was not necessary, since the test substance proved to be corrosive even without correction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8 for every exposure time.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (23.0%) and for the 60 min exposure period (14.8%) thus confirming the validity of the test system and the specific batch of tissue models.
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation in the range 20 – 100% viability between tissue replicates was < 14%.

Any other information on results incl. tables

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

Test group

Absorbance at 570 nm*

Mean absorbance of 2 tissues

Coefficient of variation (%)

Rel. absorbance (% of negative control)**

Tissue 1

Tissue 2

3 minutes treatment

Negative control

1.559

1.359

1.459

13.7

100.0

Positive control

0.359

0.311

0.335

3.3

23.0

Test substance

0.559

0.589

0.574

2.0

39.3***

60 minutes treatment

Negative control

1.337

1.452

1.394

7.9

100.0

Positive control

0.236

0.176

0.153

4.1

14.8

Test substance

0.157

0.148

0.153

0.6

10.9***

* Mean of three replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

*** Since the MTT reducing test substance extract was classified as irritant by the skin irritation test (tissue viability <50% after 3 minutes exposure), the correction procedures were not necessary.

Applicant's summary and conclusion

Interpretation of results:
other: Skin Corr. Cat. 1B (H314)
Remarks:
according to Regulation (EC) 1272/2008
Conclusions:
Under the conditions of the reconstructed human epidermis test the test substance was corrosive to skin. According to the EpiSkinTM prediction model as described in OECD TG 431 a substance which causes cell viability >= 35% after 3 min exposure and < 35% after 60 min exposure, shall be regarded as a skin corrosive Category 1B / 1C. Thus, the test substance was classified as Skin Corr. Cat. 1B according to Regulation (EC) 1272/2008.