2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

EC Number:

292-124-9

EC Name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Cas Number:

90552-04-8

Molecular formula:

C(17-19)H(32-36)O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 0023067499

Test animals / tissue source

Species:

human

Strain:

other: non-keratinized tissue construct composed of normal human-derived epidermal keratinocytes

Details on test animals or tissues and environmental conditions:

SOURCE OF RECONSTRUCTED CELLS
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Model used: EpiOcular model (OCL-200)
- Tissue batch number: 34906
- Certificate of analysis/authenticity: provided by the supplier
- Verification of tissue functionality and quality (performed by the supplier):
- Tissue viability: acceptance criteria met
- Barrier function: acceptance criteria met
- Sterility: no contamination

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl
- Concentration (if solution): undiluted test substance

Duration of treatment / exposure:

- pre-treatment with 20 μL PBS and incubated at standard culture conditions for 30 min
- 30 min of test substance treatment

Duration of post- treatment incubation (in vitro):

incubation for 12 min + 120 min at room temperature in culture medium

Number of animals or in vitro replicates:

2

Details on study design:

Two tissues were treated with each the test substance, the PC and the NC.
Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the pre-incubation medium was replaced with fresh medium, and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.
Pretreatment of the tissues
After pre-incubation, the tissues were pretreated with 20 μL PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes
Application of the test substance
Ffifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were applied concurrently with 50 μL sterile deionized water (NC) or with 50 μL
methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
Removal of the test substance and postincubation period
In order to remove the test substance, the tissues were washed with sterile PBS. For this purpose, the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immersed immediately into 12-well plates pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period).
MTT incubation
After the post-incubation period, the assay medium was replaced with 0.3 mL MTT solution, and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT incubation. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined.

EVALUATION
The test substance is considered as "non-irritant” (no UN GHS Category) if the mean relative tissue viability with a test material is greater than 60%.
A single test composed of at least two tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in case of borderline results such as non-concordant replicate measurements and/or mean percent tissue viability equal to ± 5% of the cut-off value, a second test should be considered as well as a third one in case of discordant results between the first two tests.
A “borderline“ evaluation (60 ± 5%) was determined statistically using historic data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 492.

ACCEPTANCE CRITERIA
NC: Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.8.
PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
Variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the relative difference of the viability is < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Remarks:

[%]

Value:

114.4

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance identification		Tissue 1	Tissue 2	Mean	Inter-tissue variability [%]
NC	mean OD570	1.977	1.841	1.909
	viability [% of NC]	103.5	96.5	100.0	7.1
Test substance	mean OD570	2.162	2.207	2.184
	viability [% of NC]	113.3	115.6	114.4	2.3
PC	mean OD570	0.738	0.761	0.750
	viability [% of NC]	38.7	39.9	39.3	1.2

Applicant's summary and conclusion

Conclusions:

Based on the results observed and by applying the evaluation criteria, it was concluded that 2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters does not show an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen.