Lithium iodide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 september 2021 to 14 october 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lithium iodide

EC Number:

233-822-5

EC Name:

Lithium iodide

Cas Number:

10377-51-2

Molecular formula:

ILi

IUPAC Name:

lithium iodide

Test material form:

solid

Specific details on test material used for the study:

Batch no. 1210621A-0915P

Test animals / tissue source

Species:

other: Species Bos primigenius Taurus (fresh bovine corneas)

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Industriestraße 42, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within approximately 1 hour for experiment 1 (no exact time documented in the raw data, but a transport within at least 4 hours is ensured) and 55 minutes for experiment 1b.

Test system

Vehicle:

physiological saline

Controls:

yes

Details on study design:

Preparation of Test System:

After having carefully cleaned and sterilized the cornea holders, they were kept in the incubation cham-ber at 32 ± 1 °C.
After the arrival of the corneas, they were examined visually and only corneas which were free from damages were used.
The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outer edges. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. The formation of bubbles was prevented.
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. The baseline opacity was measured by placing the cornea holder in an opacitometer and recording the illuminance (unit: LUX).
None of the corneas showed an opacity greater than seven opacity units; therefore, all corneas were used.

Description of the Method:

- Preparations
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

- Application
For each treatment group (negative control solution, test item solution or positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL of negative control solution, 750 µL of test item solution or 750 µL of positive control solution were applied to each replicate to the epithelial side of the cornea.
According to the characteristics of the controls and the test item solution, the following treatment proce-dure was performed:

- Closed Chamber Method:
The respective substance (negative control solution, test item solution or positive control solution) was applied by pipetting 750 µL of the appropriate liquid through the refill hole in the anterior holder on the cornea. The controls and the test item were given on the epithelium in a way that the cornea was evenly covered.
Exposure time of the controls and the test item on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing the anterior chambers with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chambers were filled with cMEM without phenol red and the final opacity value of each cornea was recorded.

- Permeability Test:
After the recording of the final opacity values, the cMEM without phenol red was removed from both chambers of each cornea holder. The posterior chamber, which interfaces with the endothelial side of the cornea was filled with fresh cMEM without phenol red. Then 1 mL sodium fluorescein solution was added to the front chamber of each cornea holder for the detection of permeability of the corneas.
For the controls and the test item a sodium fluorescein solution with a concentration of 5 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C in a horizontal position. After incubation, the content of each posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

IVIS

Run / experiment:

Experiment 1:

Value:

ca. 3.22

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Results 2

Irritation parameter:

cornea opacity score

Remarks:

IVIS

Run / experiment:

Experiment 1B

Value:

ca. 6.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

This in vitro study was performed to assess corneal damage potential of Lithium Iodide Anhydrous by quantitative measurements of changes in opacity and permeability in a bovine cornea. Two valid experiments were performed. The test item Lithium Iodide Anhydrous was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The test item was tested as 20% solution in HBSS. Under the conditions of this test, the test item Lithium Iodide Anhydrous showed effects on the cornea of the bovine eye. The calculated mean IVIS was 3.22 for the first experiment, IVIS was 6.18 for the experiment 1b. Therefore, the result of exp. 1 (IVIS > 3 and ≤ 55) could be verified. According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no pre-diction can be made. The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria in both experiments. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Conclusions:

According to OECD Guideline no. 437 (Jun. 2020), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no pre-diction can be made.

The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria in both experiments.

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.