Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Feb 2021 to 21 Feb 2021

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

Version / remarks:

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, (Adopted June 18, 2019).

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

EC Number:

270-470-1

EC Name:

Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Cas Number:

68441-66-7

Molecular formula:

not available due to complexity of the substance

IUPAC Name:

Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

Test material form:

liquid

Details on test material:

Identification: Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid
Batch (Lot) Number: 2019194336
Expiry date: 03 June 2022
Physical Description: Clear light yellow liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature

Additional information
Test Facility test item number: 212207/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

Information about the purity and composition of the test item is not available since the test item is an UVCB (Substance of Unknown or Variable composition, Complex Reaction Products or Biological Materials). Since a sample relevant for the purpose of this study was tested, it was concluded that the study integrity was not affected by the omission of this
information.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

Species:

human

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Test System
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 31775, Kit E.
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 49 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.7 - 36.9 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively.
Fifty μL of the undiluted test item was added into the 6-well plates on top of the tissues.

Duration of treatment / exposure:

30 ± 2 minutes at 37.0 ± 1.0 °C

Duration of post- treatment incubation (in vitro):

12 ± 2 minute immersion incubation at room temperature (Post- Soak).
After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37 °C.

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test item together with a negative control and positive control.

Details on study design:

Experimental Design
Test for the Interference of the Test Item with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed. The test item has been tested previously for possible direct MTT reduction and color interference in the Skin irritation test using EpiSkinTM as a skin model (Test Facility Study No. 20285127).
The solutions were not turned blue / purple nor a blue / purple precipitate was observed in the presence of MTT, therefore it was concluded that the test item did not interfere with the MTT endpoint.
The Optical Density (OD) for the test item solution was ≤0.08, therefore it was concluded that the test item did not interact with the MTT measurement.

Test System Set Up
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37 °C in 1.0 mL fresh pre-warmed Assay Medium. Assay Medium was supplied by MatTek Corporation, Ashland, USA.
DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent.

Test Item Preparation
No correction was made for the purity/composition of the test item.
The liquid test item was applied undiluted (50 μL) directly on top of the tissue.
Any residual volumes were discarded.

Application/Treatment of the Test Item
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-D-PBS. The tissues were incubated at standard culture conditions for a minimum of 30 minutes.
Two tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues with 50 μL Methyl Acetate (positive control) respectively.
Fifty μL of the undiluted test item was added into the 6-well plates on top of the tissues.
After the exposure period with the test item (30 ± 2 minutes at 37.0 ± 1.0 °C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.
After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a prelabeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 120 ± 15 minutes at 37 °C.

Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37 °C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol is flowing into the insert on the tissue surface.
Formazan was extracted with 2 mL of isopropanol and refrigerated for 18 ± 2 hours in the dark. At the end of the extraction period, the liquid within each insert was decanted/pipetted into the well from which it was taken.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Optical Density

Run / experiment:

Mean-Test Item

Value:

1.199

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Tissue Viability

Run / experiment:

mean - Test Item

Value:

127

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Interference of the Test Item with the MTT Endpoint
The test item was checked for possible direct MTT reduction and color interference in the Skin irritation test using EpiSkinTM as a skin model (Test Facility Study No. 20285127).
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.

Main Assay
Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 127%. Since the mean relative tissue viability for the test item was above 60% the test item is considered to be non-irritant.
The positive control had a mean cell viability after 30 ± 2 minutes exposure of 44%. The difference between the percentage of viability of two negative control tissues was ≤ 40%, which is above the maximum of 20%. The individual values of the negative control tissues (viabilities of 120% and 80%) were in the same category and were well above 60%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 492 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.5), therefore this does not affect the study integrity. The difference between the percentage of viability of two tissues treated identically with the test item was ≤ 14%, indicating that the test system functioned properly.

Any other information on results incl. tables

Mean Adsorption in the EpiOcular™ Test with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.132	0.753	0.942	±	0.268
Test item	1.132	1.267	1.199	±	0.096
Positive control	0.414	0.418	0.416	±	0.003

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background adsorption (0.0424), Isopropanol was used to measure the background adsorption.

 

Mean Tissue Viability in the EpiOcular™ Test with Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid

	Mean tissue viability (percentage of control)	Difference between two tissues (percentage)
Negative control	100	40
Test item	127	14
Positive control	44	0.5

 

Individual OD Measurements at 570 nm

	A (OD570)	B (OD570)
Negative control OD570 measurement 1 OD570 measurement 2	1.1713 1.1771	0.7982 0.7924
Test item OD570 measurement 1 OD570 measurement 2	1.1757 1.1729	1.3128 1.3063
Positive control OD570 measurement 1 OD570 measurement 2	0.4559 0.4561	0.4623 0.4591

OD = optical density

Duplicate exposures are indicated by A and B

 

Historical Control Data for EpiOcular™ Studies

	Negative control (adsorption; OD570)	Positive control (adsorption; OD570)	Positive control (viability; %)
Range	1.077 – 2.070	0.029 – 0.823	2.11 – 45.20
Mean	1.731	0.416	23.88
SD	0.225	0.209	11.49
n	54	54	54

SD = Standard deviation

n = Number of observations

The above mention historical control data range of the controls were obtained by collecting all data over the period of December 2016 to November 2020.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid. For this purpose Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid was topically applied on the Reconstructed Human EpiOcular™ Model.

 

The possible eye hazard potential of the test item was tested through topical application for 30 minutes.

 

The study procedures described in this report were based on the most recent OECD guideline.

 

Batch 2019194336 of the test item was a clear light yellow liquid. The test item was applied undiluted (50 μL) directly on top of the tissue for 30 ± 2 minutes.

 

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

 

The positive control had a mean cell viability of 44% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 492 (lower acceptance limit ≥0.8 and upper acceptance limit < 2.5). The difference between the percentage of viability of two tissues treated with test item was ≤14%, indicating that the test system functioned properly.

 

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 127%. Since the mean relative tissue viability for the test item was above 60% after 30 ± 2 minutes treatment the test item is considered to be non-irritant.

 

In conclusion, Decanoic acid, mixed esters with dipentaerythritol, octanoic acid and valeric acid is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.