1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethylene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2016 - May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

28 July 2015

Deviations:

yes

Remarks:

Tissues treated with test item were incubated at room temperature instead of at standard culture conditions (due to the volatility of the substance).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

EC Number:

615-064-0

Cas Number:

700874-87-9

Molecular formula:

C4F8O2

IUPAC Name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

Test material form:

liquid: volatile

Specific details on test material used for the study:

The test item has a boiling point of ca. 20°C and it is volatile.

- Source and lot/batch No.of test material: 150525V10
- Purity/composition correction factor: No correction factor for purity/composition applied.
- Test item storage: In refrigerator (2-8°C)
- Stability under test conditions: chemically stable.

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified 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.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

all the tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
Since the test item had a boiling point of ca. 20°C and was volatile, an excess amount of test item was applied to the tissue. During the 30 min-exposure period it was regularly checked if the skin was completely covered and if needed additional substance was added to guarantee that during the whole exposure period the tissue was covered with test item. During the exposure approximately 450 μl of test item was used per skin tissue. During the exposure the temperature of the room was approximately 20°C.

NEGATIVE CONTROL
- Amount applied: 50 µl of MilliQ water per tissue

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 50 µl Methyl Acetate per tissue

Duration of treatment / exposure:

30 ± 2 minutes

Number of animals or in vitro replicates:

Test item: 2 tissues at room temperature
Negative control (MilliQ water): 2 tissues treated at room temperature and 2 tissues treated at standard culture conditions
Positive control (Methyl Acetate): 2 tissues treated at room temperature and 2 tissues treated at standard culture conditions

Details on study design:

TEST SYSTEM
- EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23426 kit B)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes.

Interference of the test item with the MTT endpoint
- Test item was checked for possible colour intereference and direct MTT reduction before the study started

- All incubations, with the exception the exposure and post-exposure immersion at room temperature, were carried out in a controlled environment set to maintain:
- Humidity: 80 - 100%
- CO2: 5.0 ± 0.5%
- Temperature: 37.0 ± 1.0°C.

- Before the assay was started all the tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS and incubated at standard culture conditions for 30 ± 2 minutes.
- Exposure: 30 ± 2 minutes at room temperature (2 negative and 2 positive control tissues were exposed at standard culture conditions).
- Removal test item: rinsing with Ca2+Mg2+-free D-PBS (brought to room temperature)
- Post-exposure immersion: After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml Assay Medium for a 12 ± 2 minute immersion incubation at room temperature.
- Post-exposure incubation: After the Post-exposure immersion period cell culture inserts were each dried carefully and transferred to a 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. Formazan was extracted with 2 ml isopropanol refrigerated for 18 ± 2 hours in the dark. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Acceptability of the assay
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5 and the Standard Deviation value (SD) of the % viability should be ≤20%.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤20%.
c) The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.

Data evaluation and statistical procedures
A test item is considered to be positive in the in vitro eye irritation test if:
The relative mean tissue viability of two individual tissues after exposure to the test item is ≤ 60% of the mean viability of the negative controls, requiring classification for eye irritation or serious eye damage (UN GHS Category 1 or 2).
A test item is considered to be negative in the in vitro eye irritation test if:
The relative mean tissue viability of two individual tissues after exposure to the test item is > 60% of the mean viability of the negative controls, requiring no classification for eye irritation or serious eye damage (UN GHS No Category).

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Single run

Value:

99

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: None

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
The absolute mean OD570 of the negative control tissues treated at room temperature and at standard culture conditions was 1.865 and 1.773, respectively (within 0.8 and 2.5). The standard deviation value of the percentage viability of two tissues treated identically was less than 4%, indicating that the test system functioned properly.
- Acceptance criteria met for positive control: Yes.
The positive control tissues treated at standard culture condition had a viability of 41% (below 50%). The positive control tissues treated at room temperature had a viability of 51%, this result is considered acceptable considering the applied deviation from the standard procedure which requires that exposure of tissues is performed at 37 ± 1 °C. Moreover the mean viability is just above the acceptance criterion of 50% and still below 60% (positive in case of test item treatment), therefore it is considered that test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

according to EC 1272/2008

Conclusions:

Based on the evaluation of the eye hazard potential with MOVE3 using the EpiOcular cornea epithelial model performed according to OECD guideline and GLP principles, it is concluded that MOVE3 is not irritant for the eye.

Executive summary:

An evaluation of the eye hazard potential with MOVE3 using the EpiOcular™ cornea epithelial model was performed with MOVE3 according to OECD guideline 492 and GLP principles. Since MOVE3 has a boiling point of approximately 21°C an excess amount of MOVE3 was applied undiluted directly on top of the tissue at room temperature and during the 30 min-treatment period additional substance was added in order to guarantee that the tissue was covered with MOVE3 during the entire exposure period. Negative and positive controls were incubated at room temperature and at standard culture conditions with the aim of confirming the appropriateness of the test system. The positive control had a mean relative cell viability of 51% after 30±2 minutes exposure at room temperature and 41% after 30±2 minutes exposure at standard culture conditions (The mean relative cell viability of the positive control treated at room temperature is considered acceptable considering the applied deviation from the standard procedure which requires that the exposure of tissues is performed at 37 ± 1 °C and that the value is just above the acceptance criterion of 50% and still below 60%). The absolute mean OD570 of the negative control tissues was within 0.8 and 2.5 and the standard deviation value of the percentage viability of two tissues treated identically was less than 4%, indicating that the test conditions were adequate and the test system functioned properly. The relative mean tissue viability after treated with MOVE3 was 99%. Since the mean relative tissue viability for MOVE3 was above 50% after 30±2 minutes treatment MOVE3 is considered to be non-irritant and no classification is required for eye irritation or serious eye damage according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).