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Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
according to guideline
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Lithium bis[ethanedioato(2-)-κO1,κO2]difluorophosphate(1-)
EC Number:
Cas Number:
Molecular formula:
Lithium bis[ethanedioato(2-)-κO1,κO2]difluorophosphate(1-)

Test animals / tissue source

other: Bos primigenius Taurus (fresh bovine corneas)
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 1 hour and 20 minutes.

Test system

not specified
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL
Duration of treatment / exposure:
4 hours at 32 ± 1 °C
Details on study design:
1 Preparations
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
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.
After the arrival of the corneas, they were examined 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 outside. 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.
2 Experimental Parameters
Date of treatment 30. Jan. 2020
Incubation time 4 h
Negative control HBSS
Positive control imidazole, 20 % solution in HBSS
3 Method Description
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item or positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, 750 µL of the test item solution or 750 µL 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, the following treatment procedure was performed:
3.1 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 exposure time of the controls and test item solution 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.

3.2 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. 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 solution 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

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
ca. 2.47
Negative controls validity:
Irritation parameter:
in vitro irritation score
ca. 65.4
Vehicle controls validity:
Irritation parameter:
in vitro irritation score
ca. 92.88
Positive controls validity:
Other effects / acceptance of results:
In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS category I.
The test item Lithium difluorobis(oxalate)phosphate induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 65.40.

The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
This in vitro study was performed to assess corneal damage potential of Lithium difluorobis(oxalate)phosphate by quantitative measurements of changes in opacity and permeability in a bovine cornea.
The test item Lithium difluorobis(oxalate)phosphate 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 ite, opacity and permeability values were measured.

The test item was tested as a 20% solution in HBSS.
Under the conditions of this test, the test item Lithium difluorobis(oxalate)phosphate induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 65.40.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.

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

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