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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jul - 03 Aug 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted: 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142
Version / remarks:
Adopted: 31 May 2008
Deviations:
no
GLP compliance:
yes

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: 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)
- Lot number: 28855

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min exposure at room temperature, 60 min exposure at 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with phosphate buffered saline (PBS) to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- 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. The determined OD (540 - 570 nm) was 1.983 ± 0.111 (acceptance criteria: 1.0 - 3.0).
- Barrier function: According to the Supplier`s Data Sheet, the ET50 value was determined to be 5.18 h (Lower acceptance limit ET50 = 4.77 h, Upper acceptance limit ET50 = 8.72 h).
- Morphology: Tissue was inspected visually.
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected.

NUMBER OF REPLICATE TISSUES: 2 replicates for each treatment condition (3 min and 60 min experiment)

TEST FOR REDUCTION OF MTT
The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands)
solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 h at 37.0 ± 1.0ºC. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.

TEST FOR COLOR INTERFERNCE
The test substance was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the 1-h exposure. To assess the color interference, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 h at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple color change was observed.

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

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean tissue 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
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL (undiluted)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration (if solution): 8 N
Duration of treatment / exposure:
3 min and 60 min
Number of replicates:
2 replicates for each treatment condition (3 min and 60 min experiment)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min experiment
Value:
92
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min experiment
Value:
92
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- Direct-MTT reduction: The test substance did not reduce MTT directly.
- Colour interference with MTT: The test substance had no coloring potential, the solutions did not turn blue / purple nor a blue / purple precipitate was observed. It was concluded that the test item did not interfere with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range
- Acceptance criteria met for positive control: The mean relative tissue viability following 1-h exposure to the positive control was 9.3% (<15 %).
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability, the coefficient of variation between tissue replicates was ≤ 12%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 2 : Mean Tissue Viability in the in vitro Skin Corrosion Test

 

3 minute experiment, viability (% of control)

1h experiment,  

viability (% of control)

Negative control

100

100

Test substance

92

92

Positive control

9.2

9.3

Table 3: Mean Absorption in the in vitro Skin Corrosion Test

 

3 minute experiment

1h experiment

 

Mean

(OD570)

SD

Mean

(OD570)

SD

Negative control

1.858

0.036

1.775

0.047

Test substance

1.703

0.157

1.632

0.081

Positive control

0.170

0.047

0.166

0.009

 SD: Standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive according to OECD 431
Conclusions:
Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model, but no prediction on the skin irritation potential can be made and additional testing should be conducted for classification and labelling purposes.