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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:
2017-09-25 to 2017-11-03
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 July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid_paste

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended by the OECD testing guideline 431
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues
- Tissue batch number(s): Lot: 25853
- QA test date of tissue: 2017-11-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
3 min exposure at room temperature; 60 min exposure at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times with exess of DPBS
- Observable damage in the tissue due to washing: not reported


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL (1 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570)
- Wavelength: at 570 nm (OD570)
- Filter: no
- Linear OD range of spectrophotometer: no information

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: valid
- Barrier function: valid
- Morphology: valid
- Contamination: valid
- Reproducibility: valid

NUMBER OF REPLICATE TISSUES: duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue/purple colour.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. 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 [complete, e.g. 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(s) applied (volume or weight with unit): 25 ± 2mg /tissue

VEHICLE: No vihicle

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight): 50 µL deionised water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL Potassium Hydroxide
- Concentration (if solution): 8.0 N
Duration of treatment / exposure:
Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
Duration of post-treatment incubation (if applicable):
no
Number of replicates:
Duplicate EpiDermTM tissues

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes exposure
Value:
96.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non corrosive to skin
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
86
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non corrosive to skin

Any other information on results incl. tables

The test item is considered to be non-corrosive to skin:

·        since the viability after 3 minutes exposure is greater than 50% and

·        the viability after 1 hour exposure is greater than 15%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
non-corrosive to skin
Conclusions:
Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized is non corrosive to skin according to EU CLP.
Executive summary:

In an in-vitro skin irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion, RHE) (adopted July 29, 2016), Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized (100 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 minutes and 1 hour in duplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of deionised water prior to application in order to improve contact between the solid and the tissue.

Each 50 µL of negative and positive control were applied to sets of duplicate tissues, respectively.

After exposure period of 3 minutes (room temperature) and 1 hour (37 °C) the tissues were rinsed off, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. After rinsing, the formazan salt was extracted for about 20 hours at room temperature.

The test item did not reduce MTT, therefore additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The Coefficient of Variation (CV) in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the mean relative absorbance value decreased to 96.3% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 86.0%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.