Cyclooctane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-16

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU method B.69 (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:

2019-07-31

Deviations:

no

Test guideline 2

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:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclooctane

EC Number:

206-031-8

EC Name:

Cyclooctane

Cas Number:

292-64-8

Molecular formula:

C8H16

IUPAC Name:

cyclooctane

Test material form:

liquid

Test animals / tissue source

Species:

human

Strain:

other: keratinocyte strain F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method (RhCE) and considerations regarding applicability: The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- RhCE tissue used, including batch number: The EpiOcular™ Tissues (OCL-200, OCL-212) was obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia. Lot number 34914 and keratinocyte strain F1188 was used. For details on tissue quality please refer to “Any other information on materials”.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL per tissue

Duration of treatment / exposure:

30 ± 2 minutes at 37 °C and 5 % C02

Duration of post- treatment incubation (in vitro):

12 ± 2 minutes at room temperature and 120 ± 15 minutes at 37 °C and 5 % C02

Number of animals or in vitro replicates:

duplicates

Details on study design:

- Details of the test procedure used
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37 °C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37 °C and 5 % CO2 overnight (16-24 hours) without medium exchange. After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37 °C and 5 % CO2 for 30 minutes (± 2 minutes).
After the 30-minute DPBS pre-treatment, the test item, the negative and the positive control were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37 °C and 5 % CO2 for 30 ± 2 minutes.
At the end of the 30 ± 2 minutes treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (37 °C) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar " with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.
After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 12 ± 2 minutes at room temperature. After the 12 ± 2 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37 °C) assay medium for 120 ± 15 minutes at 37 °C and 5 % CO2.
- Doses of test chemical and control substances used: 50 µL per tissue
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Pre-tests for MTT-reducing capacity and colorant properties of the test item were conducted. No such properties were detected and therefore, no additional controls were needed.
- Wavelength used for quantifying MTT formazan, and linearity range of measuring device: Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany; 570 nm wavelength
- Description of the method used to quantify MTT formazan: After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37 °C and 5 % CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plate was sealed with a standard plate sealer. To extract the MTT, the plate was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissues were pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: All formulas for the calculation of the relative viability were given by the supplier of the human in vitro eye model (MatTek In Vitro Life Science Laboratories). The mean OD (optical density) of the two negative control tissues was calculated. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability was calculated according to the following formula: Viability % = (test item OD/ mean negative control OD) x 100
The prediction model according to OECD 492 was used. The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60 %. In this case no further testing in other test methods is required. If the mean percent tissue viability is less than or equal 60 %, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean. The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is two standard deviations above the current historical mean.
- Positive and negative control means and acceptance ranges based on historical data: Acceptance criterion for mean OD negative control: ≥1.088; Acceptance criterion for mean viability positive control: ≤57.9 %
- Acceptance Criteria according to OECD Guideline 492 were used. The results are acceptable if:
1. The negative control OD is >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30-minute exposure (treatment of liquid test items): below 50 % of control viability
b) 6-hour exposure (treatment of solid test items): below 50 % of control viability
3. The difference of viability between the two relating tissues of a single chemical is <20 % in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Value:

85.8

Vehicle controls validity:

not applicable

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. Please refer to "Any other information on results".
- Acceptance criteria met for positive control: Yes. Please refer to "Any other information on results".

Any other information on results incl. tables

Results of RhCE model:

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	1.999	97.9 %	2.084	102.1 %	2.042	100.0 %	2.97	4.2 %
Positive Control	0.307	15.0 %	0.323	15.8 %	0.315	15.4 %	0.57	0.8 %
Test item	1.701	83.3 %	1.802	88.2 %	1.752	85.8 %	3.46	4.9 %

 

Acceptance Criteria according to OECD Guideline 492:

	Acceptance Criterion	Result
Negative control OD	> 0.8 and < 2.5	1.999 and 2.084
Mean viability positive control	< 50 %	15.4 %
Difference of viability between the two relating tissues of a single chemical	<20 %	4.2 % (negative control) 0.8 % (positive control) 4.9 % (test item)

 

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

	Acceptance Criterion	Result
Mean OD negative control	≥1.088	2.042
Mean viability positive control	≤57.9 %	15.4 %

The study met all acceptance criteria.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not show an eye hazard potential.

Executive summary:

The objective of the present study conducted according to OECD TG 492 was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model. The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 30 ± 2 minutes. 50 µL of either the test item, the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. All acceptability criteria after treatment with the negative control and the positive control were met. Following treatment with the test item, the tissue viability was 85.8 % and, thus, higher than 60 %. Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is identified as not requiring classification and labeling according to UN GHS (No Category).