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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:
27-02-2020 to to 08-04-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2019-06-14
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Council Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008.
Version / remarks:
2008-05-20
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” (ECVAM Database Service on Alternative Methods to Animal Experimentation).
Version / remarks:
updated 2011-12 / 2012-02
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(allyloxy)ethanol
EC Number:
203-871-7
EC Name:
2-(allyloxy)ethanol
Cas Number:
111-45-5
Molecular formula:
C5H10O2
IUPAC Name:
2-(allyloxy)ethanol
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model which develops a highly differentiated and stratified epidermis after a 13-day culture period.
- Tissue batch number: 20-EKIN-010

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: With 25 mL 1 x PBS solution, residual PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in assay medium, prepared from a 3 mg/mL stock solution (in 1x PBS), 2 mL per well
- Incubation time: 3 h (± 15 min)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Approximately 50 μL test item was added to 2 mL MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours at 37±1 °C, 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. Subsequently, any observed colour changes of the solution was recorded. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (Cat. 1A) if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive to skin (Cat 1B and 1C) if the viability after 3 minutes exposure is greater than or equal to 35% and smaller than 35% after 60 minutes exposure; OR if the viability after 60 minutes exposure is greater than or equal to 35% and smaller than 35% after 240 minutes exposure.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.
- Justification for the selection of the cut-off point: The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem, 1998). The prediction model corresponds to the methods agreed by EU regulatory agencies in line with OECD 431 (OECD, 2015).
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
- Concentration: 9 g/L

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
4 h
Number of replicates:
2

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 2 replicates
Run / experiment:
1
Value:
64
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: No colour change was observed after three hours of incubation. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item is colourless, furthermore showed no ability to become coloured in contact with water. Therefore, it was considered not to be able to significantly stain the tissues and lead to false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory demonstrated technical proficiency in a separate study using the twelve Proficiency Chemicals according to OECD Test Guideline No. 431.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Acceptance criteria met for variability between replicate measurements: Yes.

Any other information on results incl. tables

Table 1: Results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells (blank mean OD: 0.0386)





























































































Substance



Optical Density (OD)



Viability (%)



∆%



Negative control: NaCl (9 g/L)


4 h exposure



1



0.791



88



23.4



2



1.000



112



mean



0.895



100



SD



0.148



16.518



CV



16.518



16.518



Positive Control: Glacial acetic acid


4 h exposure



1



0.012



1



0.9



2



0.020



2



mean



0.016



2



SD



0.005



0.608



CV



34.754



34.754



Test item:


4 h exposure



1



0.571



64



0.7



2



0.577



64



mean



0.574



64



SD



0.004



0.466



CV



0.727



0.727



 Δ%: The difference of viability between the two relating tissues

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the testing conditions. The test item can be classified as non-corrosive to skin.
Executive summary:

The EpiSkinTM test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431, adopted 14 June 2019. Disks of EPISKIN (two units / chemical / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with 1x PBS solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1 °C in an incubator with 5±1 % CO2 in a ≥ 95 % humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 64 %. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.