Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-04-27 to 2016-06-16

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2015

Deviations:

no

Test guideline 2

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” updated December 2011 / February 2012 (ECVAM Database Service on Alternative Methods to Animal Experimentation).

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

EC Number:

915-372-8

Molecular formula:

not applicable (multi-const.substance)

IUPAC Name:

Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

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: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Tissue batch number: 16-EKIN-018
- Expiry date: 09 May 2016
- Date of initiation of testing: 04 May 2016

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: approx. 25 µL, one time
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL pe rwell
- Incubation time: 3 h
- Spectrophotometer: not specified
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. In addition, the test item showed no ability to become coloured in contact with water. Thus, additional controls and data calculations were not necessary.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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 after 60 minutes exposure smaller than 35%; or if the viability after 60 minutes exposure is greater than or equal to 35% AND after 240 minutes exposure smaller than 35%.
- 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 CONTRORL
- 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

Run / experiment:

Test item

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: According to the test results of in vitro skin irritation study: No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. 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 showed no ability to become coloured in contact with water. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD value of the two negative control tissues was 1.455.
- Acceptance criteria met for positive control: yes. The positive control result showed 2 % viability. The difference of viability between the two tissue replicates:
Negative control: 0 %
Positive control: 1 %
Test item: 0 %
- Acceptance criteria met for variability between replicate measurements: All validity criteria were within acceptable limits in the experiment therefore the study can be considered as valid.

Applicant's summary and conclusion

Interpretation of results:

other: not classified as corrosive

Conclusions:

In conclusion, in this in vitro skin corrosion test using the EPISKIN model with the test item the results indicated that the test item is not corrosive to skin. In conclusion, according to the UN GHS classification the test item can be classified as Non-corrosive, under the utilised testing conditions. However, further data is required in order to predict skin irritating properties.

Executive summary:

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of EPISKIN (two units) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1 x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 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 four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 100 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.