1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 24, 2018 - April 26, 2018

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

Test guideline 1

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:

2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT

Version / remarks:

2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: 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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

EC Number:

944-870-8

Molecular formula:

C18H14F20O9S3

IUPAC Name:

1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 mg

Duration of treatment / exposure:

6 hours (± 15 minutes)

Duration of post- treatment incubation (in vitro):

18 hours (± 15 minutes)

Number of animals or in vitro replicates:

The test item as well as the positive and negative control were tested in batch-duplicates. Therefore, a total number of six tissues was used in this study.

Details on study design:

- RhCE tissue construct used, including batch number
The EpiOcular™ Tissues (OCL-200, OCL-212) (Lot No: 27036) was obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.

- Doses of test chemical and control substances used
Solid test item: 50 mg per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

- Duration and temperature of exposure
incubated at 37°C and 5% CO2 for 6 hours (± 15 minutes)
At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes (± 2 minutes) at room temperature. After the 25 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 18 hours (± 15 minutes) at 37°C and 5% CO2.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals
The non-colored test item was tested for its ability to become colorant after contact with water or isopropanol. For this purpose, 50 mg of the test item were added to 1.0 mL of water in a 6-well plate and the mixture was incubated for 1 hour at 37 ± 1°C and 5% CO2 protected from light. Furthermore, 50 mg were added to 2 mL isopropanol, the same amount as used for MTT extraction, and was incubated in 6-well plates for 2 to 3 hours at room temperature.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
The test item as well as the positive and negative control were tested in batch-duplicates. Therefore, a total number of six tissues was used in this study.

- 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 6-well plate containing 2 ml isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
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
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.

- Acceptable Criteria
The results are acceptable if:
1. The negative control OD >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50% of control viability
b) 6 hour exposure: 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:

other: % viability

Run / experiment:

1st run

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table 1: Results

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	1.992	97.9%	2.076	102.1%	2.034	100.0%	2.97	4.2%
Positive Control	0.515	25.3%	0.690	33.9%	0.603	29.6%	6.08	8.6%
Test item	0.021	1.0%	0.022	1.1%	0.022	1.1%	0.07	0.1%

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made; further testing with other test methods is required because RhCE test methods cannot resolve between UN GHS Categories 1 and 2

Conclusions:

Following treatment with the test item, the tissue viability was 1.1% and, thus, lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model according to OECD TG 492. 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 6 hours (± 15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 2.034 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 29.6% (study acceptance criterion: <50%). Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was 1.1% and, thus, lower than 60%,i.e.according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted and further testing is necessary.