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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May 2017 - 05 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
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:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 03 November 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: white to off white powder or flakes
Storage conditions: at room temperature protected from light
Specific details on test material used for the study:
- Correction factor for purity: no correction required
- Stability at higher temperatures: yes, maximum temperature: 60°C for a maximum duration of 1 hour

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
TEST SYSTEM:
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.
- Justification: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
Tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free- DPBS.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: at least 50 mg

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

POSITIVE CONTROL
Amount applied: 50 µl Methyl Acetate per tissue
Duration of treatment / exposure:
6 hours ± 15 minutes
Duration of post- treatment incubation (in vitro):
18 hours (after a post-soak period of 25 ± 2 minutes)
Number of animals or in vitro replicates:
Test item: 2 tissues
Negative control (MilliQ water): 2 tissues
Positive control (Methyl Acetate): 2 tissues
Details on study design:
TEST SYSTEM
- EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23479)
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 by adding the test item to MTT medium.

- All incubations, with the exception the exposure and post-exposure immersion at room temperature, were carried out in a controlled environment:
- Humidity: 46 - 83%
- CO2: 5.0 ± 0.5%
- Temperature: 36.4 - 37.2°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: 6 hours ± 15 minutes at 37.0 ± 1.0°C
- 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 of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak).
- Post-exposure incubation: After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 18 hours ± 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 for 2 - 3 hours at room temperature with gentle shaking. 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.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

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.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.

Data evaluation:
* The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
* The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.

Results and discussion

In vitro

Results
Irritation parameter:
other: Mean tissue viability (% of negative control)
Run / experiment:
Single run
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
SD: 7.9%
Positive controls validity:
valid
Remarks:
29% (SD: 6.8%)
Remarks on result:
other: SD: 2.1%
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD570 measurements of the negative control tissues were between 0.8 and 2.5 (i.e., a mean of 1.586 ± 0.125). The standard deviation value of the percentage viability of two tissues treated identically was 7.9%, indicating that the test system functioned properly.
- Acceptance criteria met for positive control: Yes, the positive control tissues had a mean tissue viability <50% (i.e., 29%).
- The standard deviation from individual % tissue viabilities of the two identically treated replicates was <20% (i.e., 2.1%).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
Based on the evaluation of the eye hazard potential with Ethylene glycol dibenzoate using the EpiOcular cornea epithelial model and performed according to OECD guideline and GLP principles, it is concluded that Ethylene glycol dibenzoate is not irritant for the eye.