Linseed oil, ester with pentaerythritol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 24, 2017 to April 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

adopted July 26, 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Linseed oil, ester with pentaerythritol

EC Number:

271-985-4

EC Name:

Linseed oil, ester with pentaerythritol

Cas Number:

68648-28-2

Molecular formula:

UVCB

IUPAC Name:

2,3-dihydroxypropyl (7E,10E,13E)-hexadeca-7,10,13-trienoate; bis(3-[(7E,10E,13E)-hexadeca-7,10,13-trienoyloxy]-2-hydroxypropyl (9E)-octadec-9-enoate); 3-hydroxy-2-(hydroxymethyl)-2-[(octadec-9-enoyloxy)methyl]propyl (9E,12E,15E)-octadeca-9,12,15-trienoate; 3-hydroxy-2-{[(9E)-octadec-9-enoyloxy]methyl}-2-{[(9E,12E)-octadeca-9,12-dienoyloxy]methyl}propyl (9E,12E,15E)-octadeca-9,12,15-trienoate

Test material form:

liquid

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cul-tured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL unchanged

Duration of treatment / exposure:

28 minutes

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

Duplicate

Details on study design:

Pre-tests:

Assessment of direct reduction of MTT by the test substance: The test substance was tested for the ability of direct formazan reduction. To test for this ability, 50 µL of the liquid test substance were added to 1 mL of MTT reagent in a 6-well plate and the mixture was incubated in the dark at 37±1°C, 5.0±1% CO2 and 80–100% relative humidity for 3 h. 1 mL of MTT reagent plus 50 µL of deminerelised water was used as negative control. The MTT reagent did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Assessment of coloured or staining test substances: It was tested whether the test substance develops a colour without MTT addition. 50 µL of the test substance was added to 2 mL isopropanol, incubated in 6-well plates on an or-bital shaker for 2 h at room temperature. Then, two 200 µL aliquots of the resulting solu-tion and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of OD for isopropanol, the OD of the test substance solution was -0.002 (≤ 0.08). Therefore, the main test was performed without colourant controls.

Main test:

Preparations: On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent directly before use. The assay medium was warmed in the water bath to 37±1°C. 6-well-plates were labelled with test substance, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All 24 inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37±1°C, 5±1% CO2 and 80–100% relative humidity for 1 h. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37±1°C, 5±1% CO2 and 80–100% relative humidity for 16 h and 30 minutes.

Exposition and post-treatment: After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and then incubated at 37±1°C, 5±1% CO2 and 80–100% relative humidity for 30 minutes. After that, 50 µL of the controls and the test substance were applied in duplicate in 1- minute-intervals. This was done in such a manner that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 28 minutes at 37±1°C, 5±1% CO2 and 80–100% relative humidity. At the end of the exposure time, the inserts were removed from the plates in 1 minute in-tervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature. After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment in-cubation, the tissues were incubated for 120 minutes at 37±1°C, 5±1% CO2 and 80–100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

MTT assay and extraction: A 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solu-tion. The plate was incubated for 180 minutes at 37±1°C, 5±1% CO2 and 80 – 100% relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then stored in the refrigerator overnight. On the next day the plate was shaken on an orbital shaker for 2 h at room temperature, protected from light.

Measurement: After 2 h, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were also pipetted, serving as a blank. The plate was read in a plate spectrophotometer at 570 nm.

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability

Run / experiment:

Tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls

Value:

ca. 105.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.7 (> 0.8 and < 2.5). The positive control induced a decrease in the relative absorbance as compared to the negative control to 38.4%. Variation within the replicates was acceptable (< 20%). The related infromation on results were mentioend in table in below section 'any other information on results incl. tables'.

Any other information on results incl. tables

Absorbance values blank isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.038	0.038	0.037	0.038	0.038	0.039	0.038	0.039	0.038

Absorbance values negative control, positive control and test Item (OD at 570 nm) :

Designation	Measurement	Negative Control	Positive Control	linseed oil, ester with pentaerythritol
Tissue 1	1	1.746	0.711	1.819
	2	1.698	0.694	1.811
Tissue 2	1	1.729	0.677	1.829
	2	1.707	0.655	1.783

Mean absorbance negative control, positive control and test Item:

Designation	Negative Control	Positive Control	linseed oil, ester with pentaerythritol
Mean – blank (Tissue 1)	1.684	0.665	1.777
Mean – blank (Tissue 2)	1.680	0.628	1.768

% Viability positive control and test Item :

Designation	Positive Control	linseed oil, ester with pentaerythritol
% Viability (Tissue 1)	39.5%	105.6%
% Viability (Tissue 2)	37.3%	105.1%
% Viability Mean	38.4%	105.4%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was identified as non-irritating to eyes.

Executive summary:

A study was conducted to determine eye irritation potential of the test substance in the reconstructed human Cornea-like Epithelium according to OECD Guideline 492, in compliance with GLP. Before conducting the main study, the substance was tested for ability of direct formazan reduction The MTT reagent did not change its colour, therefore direct MTT reduction had not taken place. Duplicate tissues of the reconstructed human Cornea-like Epithelium were treated with 50µL of test substance for 28 minutes. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD >0.8 and <2.5, OD was 1.7. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 38.4% (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test substance, the mean value of tissue viability was 105.4%. This value is well above the threshold for eye irritation (≤60%). Under the study conditions, the test substance was identified as non-irritating to eyes (Andres, 2017).