(Z)‐ethyl 2‐methylpent‐3‐enoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2020 - 18 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Version / remarks:

June 2019

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

Details on animal used as source of test system:

EpiDerm Skin Model (EPI-200, Lot no.: 33783 Kit F)

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- All cells used to produce Epiderm™ are purchased or derived from tissue obtained by MatTek
Corporation from accredited institutions.
- Cells are screened for potential biological contaminants (HIV-1, Hepatitis B, Hepatitis C, bacteria, yeast and fungi)
- Surface: 0.6 cm²
- The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment at 37.0 ± 1.0°C (actual range 35.9 - 37.3°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2 replicates per exposure duration, two negative controls per exposure duration and two positive controls per exposure duration

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8).
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

INTERPRETATION (See table 1)
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Fifty μL of the undiluted test item was added into the 6-well plates on top of the skin tissues.

Duration of treatment / exposure:

3 minutes or 1 hour

Number of replicates:

2 replicates per exposure duration

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute treatment

Value:

98

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour treatment

Value:

65

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT INTERFERENCE
- The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0027 and 0.0001, respectively. Therefore it was concluded that the test item did not induce color interference.
In addition, because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint.

RESULTS
- Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 98% and 65% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.

ACCEPTABILITY OF RESULTS
- 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. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.2%.
- In the range of 20 - 100% viability the Coefficient of Variation between replicates of the negative control tissues was ≤ 14%, indicating that the test system functioned properly.
- The Coefficient of Variation between replicates of the test item tissues was 33% for the 1-hour procedure. Although this value exceeds the acceptability criteria of 30%, all individual viabilities were in the same classification category, therefore the test outcome was considered to be valid.

Any other information on results incl. tables

Table 2. Mean Absorption in the in vitro Skin Corrosion Test with Ethyl 2-methyl-3-pentenoate

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.784	1.776	1.780	±	0.006	1.863	1.600	1.732	±	0.186
Test item	1.647	1.832	1.739	±	0.131	1.340	0.898	1.119	±	0.313
Positive control	0.111	0.130	0.120	±	0.013	0.144	0.105	0.125	±	0.027

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In table 1 the values are corrected for background absorption (0.0434). Isopropanol was used to measure the background absorption.

 

Table 3. Mean Tissue Viability in the in vitro Skin Corrosion Test with Ethyl 2-methyl-3-pentenoate

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Test item	98	65
Positive control	6.8	7.2

 

Table 4. Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	0.4	14
Test item	10	33
Positive control	14	27

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The substance is not corrosive to the skin.

Executive summary:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model) performed according to OECD TG 431 and GLP principles, the influence of the test substance on the viability of human skin was tested. The test item was applied undiluted (50 μL) was applied directly on top of the skin tissue. The positive control had a mean relative tissue viability of 7.2% after the 1-hour exposure.
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. In the range of 20 - 100% viability the Coefficient of Variation between replicates of the negative control tissues was ≤ 14%, indicating that the test system functioned properly.
The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 98% and 65%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.

In conclusion, the test substance is not corrosive to the skin in accordance with EU CLP (EC no1272/2008 and its amendments).