Fatty acids, C18-36, esters with ethylene glycol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 September 2017 to 09 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-36, esters with ethylene glycol

EC Number:

305-673-7

EC Name:

Fatty acids, C18-36, esters with ethylene glycol

Cas Number:

94944-95-3

Molecular formula:

C40H78O4/C70H138O4

IUPAC Name:

Fatty acids, C18-36, esters with ethylene glycol

Test material form:

solid

Test animals / tissue source

Species:

other: human keratinocytes

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia).

Lot No.: 27012
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free D-PBS Dulbecco´s Phosphate Buffered Saline

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant solution (Isopropanol) For extraction of formazan crystals

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL MTT
- Incubation time: 3 h
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1)

NUMBER OF REPLICATE TISSUES: 2/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control

METHOD OF CALCULATION
viability [%] = 100* (ODti or pc/meanODnc)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA
If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is labeled irritant.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg test substance, 50 uL controls

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes in Assay Medium (to remove any test item absorbed into the tissue) and 18 hours in Assay Medium at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

Number of animals or in vitro replicates:

2 replicates per treatment (1 for blank)

Details on study design:

At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues (3 times) with Ca++Mg++-free DPBS (brought to room temperature).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 25 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm Assay Medium. The tissues are incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that no isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Results and discussion

In vitro

Results

Irritation parameter:

other: viability %

Value:

92.2

Negative controls validity:

valid

Remarks:

OD 1.484 and 1.530

Positive controls validity:

valid

Remarks:

mean rel viability 28.6%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

No MTT interefernce was observed, no color interference was found

Any other information on results incl. tables

Dose Group	OD570 Well 1	OD570 Well 2	Mean OD (Tissue 1/2)	Mean OD Tissue 1 and 2 corrected	Mean OD of 2 Tissues Blank corrected	Rel. Viability [%] Tissue 1 and 2*	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Viability [%]**
Blank	0.035	0.034	0.035
Negative Control	1.543	1.494	1.518	1.484	1.507	98.5	3.0	100.0
	1.569	1.560	1.564	1.530		101.5
Positive Control	0.493	0.479	0.486	0.451	0.431	30.0	2.7	28.6
	0.446	0.446	0.446	0.411		27.3
Test Item	1.425	1.429	1.427	1.392	1.507	92.4	0.4	92.2
	1.412	1.429	1.420	1.385		92.0

* Relative viability [rounded values]: 100*(absorbance/absorbance neg contr)

** Mean relative viability [rounded values]: 100 * (mean absorbace)/mean absorbance negative control

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eyes

Executive summary:

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test. The substance interfered with MTT reduction, but not with color.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 6 hours.

Irritating effects were not observed following incubation with the substance. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (92.2%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance does not possess any eye irritating potential.