[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Oct to 06 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

adopted in 2016

Deviations:

yes

Remarks:

no information on demonstration of proficiency

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, vegetable-oil, polymd., esters with 1,2-hexadecanediol

Cas Number:

68910-99-6

Molecular formula:

Not applicable (i.e., UVCB substance)

IUPAC Name:

Fatty acids, vegetable-oil, polymd., esters with 1,2-hexadecanediol

Test material form:

other: paste

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM; reconstructed three-dimensional human epidermis (EPI-200)

Justification for test system used:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

This system is recommended in international guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200, MatTek Corporation, Ashland MA, U.S.A.)
- Tissue batch number: 27144
- Delivery date: 01 OCT 2017
- Date of initiation of testing: 02 Oct 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min exposure), 37.0 ± 1.0 C (60 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

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 1.989 ± 0.122 (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 6.21h (acceptance criteria: 4.77 - 8.72 h).
- Contamination: The cells used to produce the EpiDermTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: test item: 2 x 2 replicates (3 and 60 min); negative and positive controls: 1 replicate each (3 and 60 min)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- not applicable

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

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

Amount/concentration applied:

TEST MATERIAL
- Amount applied: An excessive amount of the paste test item was applied directly on top of the skin tissue using a spatula. The test item was spread to match the size of the tissue.

VEHICLE
- not applicable

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

test item: 2 x 2 replicates (3 and 60 min); negative and positive controls: 1 replicate each (3 and 60 min)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

79

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of corrosion

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not show reducing capacity 1 hour after MTT incubation.
- Colour interference with MTT: The test substance did not change colour, when mixed with Milli-Q water and thus passed the colour interference pre-test.

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 viability of the tissue replicates treated with the positive control for 1 hour, is < 15% compared to the negative control (7.4%).
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 30% (≤ 16%) indicating that the test system functioned properly.

Any other information on results incl. tables

Table 2. Results after 3 and 60 min exposure with the test substance and controls

	3-minute exposure					1-hour exposure
	A (OD570)	B (OD570)	Mean		SD	A (OD570)	B (OD570)	Mean		SD
Negative control	1.810	2.167	1.988	±	0.253	1.822	1.893	1.857	±	0.050
Test item	1.608	1.528	1.568	±	0.057	1.628	1.745	1.686	±	0.083
Positive control	0.508	0.158	0.333	±	0.247	0.156	0.117	0.137	±	0.028

SD = Standard deviation

Duplicate exposures are indicated by A and B.

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 was conducted for classification and labeling purposes.