Gallium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01/04/2019 - 04/04/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Gallium

EC Number:

231-163-8

EC Name:

Gallium

Cas Number:

7440-55-3

Molecular formula:

Ga

IUPAC Name:

gallium

Test material form:

solid: bulk

Specific details on test material used for the study:

Batch no 1047000052

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cor-nea. It consists of highly organized basal cells. These cells are not transformed or trans-fected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 02. Apr. 2019
Batch no.: 27098

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

28 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2 replicates

Details on study design:

Preparations

On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use.
The assay medium was warmed in the water bath to 37 ± 1°C.
6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 16 hours and 59 minutes.

Exposure and Post-Treatment

After overnight incubation, the tissues were pre-wetted with 20 μL DPBS and then incu-bated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 30 minutes. After that, 50 μL of the controls and 50 μL of the test item were applied in duplicate in one- minute-intervals. This was done in such a way that the epithelial surface of the tissue was uniform-ly covered. At the beginning of the experiment (application of negative controls), a stop watch was started. After dosing the last tissue of each plate, the plate was transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At the end of the exposure time, the inserts were removed from the plates in one- minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature.
After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment incu-bation, the tissues were incubated for 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
After the post-treatment incubation, the MTT assay was performed.

MTT Assay and Extraction

A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solu-tion. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% rela-tive humidity.
At last, each insert was thoroughly dried and set into the empty 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was sealed to avoid evaporation of the solvent and stored in the refrigerator over-night. On the next day, the plate was shaken (120 rpm) for 2 hours at room temperature, protected from light.

Measurement

The inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: % tissue viability

Run / experiment:

1

Value:

99.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: % tissue viability

Run / experiment:

2

Value:

99.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, gallium is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

Under the conditions of the test, gallium is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

After treatment with the test item, the mean value of relative tissue viability was reduced to 99.2 %. This value is above the threshold for eye irritation potential (≤ 60%).

All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.7 (> 0.8 and < 2.5).

The positive control induced a decrease in tissue viability as compared to the negative control to 43.5%. Variation within the replicates of the controls and the test item was acceptable (< 20%).

For these reasons, the result of the test is considered valid.