ETHYL 2-METHYL-4-PENTANOATEETH OXANOATE-369 CRUDE

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February 2021 - 15 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Substance information is used to read across from.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(Z)‐ethyl 2‐methylpent‐3‐enoate

EC Number:

854-058-4

Cas Number:

58625-89-1

Molecular formula:

C8H14O2

IUPAC Name:

(Z)‐ethyl 2‐methylpent‐3‐enoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC). The test item was not corrosive in the EpiDerm model.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): 21-EKIN-006
- Expiration date: 15 February 2021
- Blood of the donors has been verified to be absent of HIV1 and 2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs, bacteria, fungus and mycoplasma.

ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 78 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.1°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES: 1

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit 1.5) and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

INTERPRETATION
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL

NEGATIVE CONTROL
- Amount applied: 25 µL

POSITIVE CONTROL
- Amount applied: 25 µL

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

3 hours at 37.0 °C

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

94

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT REDUCTION AND COLOR INTERFERENCE
The test item was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model as a skin model (Test Facility Study No. 20275716). Because the solutions did not turn blue / purple, nor a blue / purple precipitate was observed and the OD for the test item solution was ≤0.08, it was concluded that the test item did not interfere with the MTT endpoint.

RESULTS
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 94%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

CONTROLS
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 5.4%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 4.2%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 2. Mean Absorption in the In Vitro Skin Irritation Test with Ethyl 2-methyl-3-pentenoate

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.263	1.304	1.200	1.256	±	0.052
Test item	1.183	1.216	1.156	1.185	±	0.030
Positive control	0.053	0.092	0.059	0.068	±	0.021

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.0431). Isopropanol was used to measure the background absorption.

 

Table 3. Mean Tissue Viability in the In Vitro Skin Irritation Test with Ethyl 2-methyl-3-pentenoate

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	4.2
Test item	94	2.4
Positive control	5.4	1.7

 

Applicant's summary and conclusion

Interpretation of results:

other: Not skin irritant in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance does not cause skin irritation.

Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested. The test item was applied undiluted (25 μL), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 ± 1 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. 

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 94%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.
The positive control had a mean cell viability of 5.4% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 4.2%, indicating that the test system functioned properly.

In conclusion, the test substance is non-irritant in the in vitro skin irritation test.