Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2017 - 30 June 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white powder with lumps
- Test item storage: at room temperature protected from light
Specific details on test material used for the study:
- pH (1% in water, indicative range): 5.12 - 4.78

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)
Controls:
yes, concurrent positive control
yes, concurrent negative control
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 (Triton X (0.3%)): 2 tissues
Details on study design:
TEST SYSTEM
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 23487)
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes.

- Doses of test chemical and control substances used: 54.8 to 58.6 mg of test substance, 50 μL of the positive control and 50 μL of the negative control.
- 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.

- Justification for the use of a different positive control than neat methyl acetate: The positive control (Methyl Acetate) was not supplied with the kit. Therefore Triton X (0.3%) was used as a positive control.Triton X 0.3% shows a response which could be expected based on the ET-50 (exposure time required to reduce tissue viability by 50%) of this kit which was 29.81 minutes, therefore this does not affect the study outcome.

Interference of the test item with the MTT endpoint
- Test item was checked for possible colour intereference before the study started by adding 50 mg of the test item to 1.0 mL Milli-Q water and incubated for 1 hour. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
- For MTT reduction 50 mg of the test item was added to 1mL MTT solution. This mixture was incubated for 3 hours. After incubation, the mixture was checked for a blue/purple colour.

- All incubations, with the exception the exposure and post-exposure immersion at room temperature, were carried out in a controlled environment:
- Humidity: 77-91%
- CO2: 5.0 ± 0.5%
- Temperature: 36.9 - 37.5°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.

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%.-
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%.

Results and discussion

In vitro

Results
Irritation parameter:
other: Mean tissue viability (% of negative control)
Run / experiment:
Single run
Value:
101
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean tissue viability: 100%
Positive controls validity:
valid
Remarks:
Mean tissue viability: 1.87%
Other effects / acceptance of results:
OTHER EFFECTS:
- Colour interference of the test item: no
- Reduction of MTT by the test item: no
- 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 1.574 ± 0.025), which was in the historical control data range.
- Acceptance criteria met for positive control: Yes, the positive control tissues had a mean tissue viability <50% (i.e., 1.87%).
- The standard deviation from individual % tissue viabilities of the two identically treated replicates was <20% (i.e., <2%).

Any other information on results incl. tables

Table 1 Historical control data for EpiOcular™ Studies

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

1.228 – 2.090

0.320 – 0.535

17.80 – 34.18

Mean

1.53

0.42

27.55

SD

0.23

0.07

5.31

n

12

12

12

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2013 to May 2017.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
Based on the evaluation of the eye hazard potential with ROC-118 using the EpiOcular cornea epithelial model and performed according to OECD guideline and GLP principles, it is concluded that ROC-118 is not irritant for the eye. The substance is not classified according to GHS and according to Regulation (EC) No. 1272/2008.
Executive summary:

An in vitro eye irritation study was performed according to OECD guideline and GLP principles to test the eye hazard potential of ROC-118. The substance was directly applied on top of the Reconstructed Human EpiOcular model for 6 hours ± 15 minutes at an amount of 54.8 -58.6 mg. After the exposure period, the tissues were rinsed and incubated for an additional 18 hours prior to determination of the cytotoxic effect. The relative mean tissue viability after 6 hours ± 15 minutes treatment with the test substance, compared to the negative control, was 101%. The acceptability criteria for the positive and negative control were met. Based on these results, ROC-118 is considered to be non-irritant to the eyes and is not classified according to GHS and according to Regulation (EC) No. 1272/2008.