Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 July - 05 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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 in 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP - Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human-derived epidermal keratinocytes (NHEK), Strain 00267
Justification for test system used:
The EpiDerm(TM) Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and has been used for in vitro experiments for many years. It is known for its similarity to human skin.
Vehicle:
unchanged (no vehicle)
Remarks:
moistened with water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm(TM) Skin Model
- Tissue batch number(s): 28646 and 28658

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After 3 or 60 min of application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate“ containing 300 μL pre-warmed assay medium per well. All inserts were treated in the same manner.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm(TM) tissues were assesed by an MTT cell viability test. The determined OD (540-570 mm) was 2.216 ± 0.075 for the first experiment and 1.802 ± 0.137 for the second experiment (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 h for the first and second experiment (acceptance criteria: 4.77 - 8.72h)
- Morphology: not reported
- Contamination: The cells used to produce the EpiDerm(TM) tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected
- Reproducibility: The results of the positive and negative controls showed reproducibility over time.

Since the test substance did not directly reduce the MTT solution, an additional functional check was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 independent experiments were performed

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%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
-
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25 mg + 25 µL distilled water

NEGATIVE CONTROL
- Amount(s) applied: 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied: 50 µL 8 N KOH
Duration of treatment / exposure:
3 and 60 min
Duration of post-treatment incubation (if applicable):
3 h with MTT solution
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure (1st experiment)
Value:
90.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean value of two tissues
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure (1st experiment)
Value:
17.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean value of two tissues
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure (2nd experiment)
Value:
93.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean value of two tissues
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure (2nd experiment)
Value:
5.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean value of two tissues
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes: The mena OD of the tissue replicates treated with the negative control was >= 0.8 and <= 2.8 for every exposure time (values between 1.680 and 1.723).
- Acceptance criteria met for positive control: yes: The mean relative tissue viability (% negative control) of the positive control was < 15% (6.3%) after 60 minute treatment.
- Acceptance criteria met for variability between replicate measurements: yes: The coefficient of variance (CV) (in the range 20 - 100%) of replicate tissues of all dose groups was <= 30% (values between 3.1% and 12.0%).

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS Category 1B (H314) according to Regulation (EC) No 1272/2008
Conclusions:
In this study under the given conditions the test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment but not below 50% after 3 min treatment. The test item is therefore classified as “corrosive“ with sub-category 1B.