Ethyl decanoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10 November 2017 to 6 December 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

Qualifier:

equivalent or similar 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

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl decanoate

EC Number:

203-761-9

EC Name:

Ethyl decanoate

Cas Number:

110-38-3

Molecular formula:

C12H24O2

IUPAC Name:

ethyl decanoate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. 16090585
- Expiration date of the lot/batch: 19 September 2018
- Purity test date: 22 September 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions:at least 6 months from the manufacture date
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Pure

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30±2 µL (60 µL/cm2)
- Concentration (if solution): pure

VEHICLE
No vehicle was used

Duration of treatment / exposure:

30 ± 2 minutes

Duration of post- treatment incubation (in vitro):

30 ± 2 minutes

Number of animals or in vitro replicates:

Duplicates were used for each condition

Details on study design:

- Details of the test procedure used
Prior to the application of the test item, HCE tissues are transferred into 24-well plates filled with EPISKIN maintenance medium. 30µL (±2µL) of test material and/or controls are topically applied on the surface of HCE tissues (0.5cm2) Tissues treated with test item and with control substances tested concurrently to test item are incubated for 30 minutes (±2) at standard culture conditions.
After exposure to the test material to the test material, the treated tissues are rinsed with PBS and transferred into 24 well plates filled with maintenance medium. This rinsing step is followed by a 30 minutes(±2) post exposure immersion in maintenance medium at standard culture conditions, prior to performing the MT assay.
In order to evaluate the tissue viabilit, MTT assay was performed. The inserts are transferred to a 24 well plate containing 300 µL of MTT solution at 1mg/ml in maintenance medium. After 180 minutes, incubation in standard culture conditions, the precipitated blue formazan is then extracted from the tissues using isopropanol. Tissues tested with liquid test items are extracted from both the top and the bottom of the tissues, while tissues with colourd liquids are extracted from the bottom of the tissue only.

- RhCE tissue construct used, including batch number HCE SkinEthicTM ; HCE/S/5 lot 17-HCE-058

- Doses of test chemical and control substances used 30 µL of pure test chemical, positive and negative control were used.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
30 minutes exposure period at 37°C ; post exposure immersion 30 minutes at 37°C ; 180 minutes of MTT incubation period

- Justification for the use of a different negative control than ultrapure H2O (if applicable) not applicable. The PBS was required according to the guideline (OECD TG 49 2)

- Justification for the use of a different positive control than neat methyl acetate (if applicable) methyl acetate was used

- Description of any modifications to the test procedure not applicable

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
Direct MTT reduction controls : 30µL of the test material was added to 300 µL of 1 mg/mL MTT solution and the mixture is incubated for 3 hours at standard culture conditions.
Colour Interference : 10 µL of test material are mixed with 90 µL of distilled water. The solution is incubated for 30 minutes at room temperature

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
duplicates were used

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
570 nm

- Description of the method used to quantify MTT formazan
The optical densities OD from individual replicate tissues are first corrected by substracting the blank mean OD value. The mean from corrected OD values (ODcorr) is calculated in each experimental group.
For normal condition, the percentage of tissue viability (%viab) is calculated from the mean ODcorr values relative to the mean viability of negative control (PBS treated epithelium)
%Viab [Treated] = ([Treated]ODcorr mean / [Negative Control]ODcorr mean) x100

- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable) not applicable

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The eye irritant potential of the test item is predicted by the mean percent tissue viability (mean between 2 tissue replicates) normalised to the negative control which is set at 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items according to UN-GHS is 60% (higher than 60% of viability : not classified ; lower than 60% of viability : no prediction can be made)

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
not specified

- Complete supporting information for the specific RhCE tissue construct used
Only batch and RhCE tissue was given

- Reference to historical data of the RhCE tissue construct
not specified

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
the methyl acetate showed positive responce and the capability of the test system to measure a decrease of cellular viability.

- Positive and negative control means and acceptance ranges based on historical data
The mean viability of tissues treated with the positive control (methyl acetate n=2) is =< 30%.
The mean OD of the negative control (NC n=2) is >= 1.4 with an upper acceptance limit of =<2.5

- Acceptable variability between tissue replicates for positive and negative controls
Conform
- Acceptable variability between tissue replicates for the test chemical
Conform

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

Maint test - Negative Control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

Main test - Positive Control

Value:

2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

% tissue viability

Run / experiment:

Main test - Test Item

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The methyl acetate was used as positive control and showed potency of the test system to measured tissue viability

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: conform
- Acceptance criteria met for positive control: conform
- Range of historical values if different from the ones specified in the test guideline: not applicable

Any other information on results incl. tables

Table 1 :Results

Condtion	Optical Density Mean	Cellular viability
Negativecontrol	1.759 ± 0.011	100
Positive control	0.041 ± 0.001	2
Test item	1.766 ± 0.017	100

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the test item ethyl caprate, did not induced decrease of tissue viability. Hence, according to the CLP criteria, the caprate was not classified for eye irritation.

Executive summary:

This GLP compliant in vitro study was performed in order to assess the potential eye irritation potential of the test item, ethyl caprate. This study was performed according to the OECD TG 492 method.

HCE SkinEthicTM model was used as test system in this study. 30 µL of ethyl caprate was applied on the EpiSkinTM for 30 minutes. Buffered saline solution (PBS) was used as negative control and methyl acetate was used as positive control. Duplicates were used for each condition. The 30 minutes exposure period was followed by a 30 minutes post incubation period. After this step, the viability of the HCE SkinEthicTM model was measured by a MTT assay.

The postive control induced a decrease of cellular viability (2% of viability). The negative control did not induced a viability decrease. The viability of the test system was 100% when exposed to the test item.

The acceptance criteria of the assay was considered as validated.

Based on the results of this study, the test item ethyl caprate, did not induced decrease of tissue viability. Hence, according to the CLP criteria, the caprate was not classified for eye irritation.