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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 guideline
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 09. Oct. 2017
GLP compliance:
yes (incl. QA statement)

Test material

Chemical structure
Reference substance name:
Ammonium hexafluorozirconate
EC Number:
EC Name:
Ammonium hexafluorozirconate
Cas Number:
Molecular formula:
ammonium hexafluorozirconate
Test material form:
solid: particulate/powder

Test animals / tissue source

other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
- Source: slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): the cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were transported to the test facility in Hanks' Balanced Salt Solution with 1% Penicillin-Streptomycin solution in a suitable cooled container wihtin 1h and 30 minutes.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was tested neat. The surface of the corneas was completely and evenly covered. The amounts of the test item used where as follows:
replicate 1: 543.7 mg; replicate 2: 557.4 mg; replicate 3: 553.8 mg.
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 replicates
Details on study design:
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.
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 and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, a defined amount of test item and 750 μL positive control solution were applied to each replicate to the epithelial side of the cornea.
After the recording of the final opacity value, the cMEM without phenol red was removed from both chambers. 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 for the detection of permeability of the corneas. For solid non-surfactant test items, 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 the posterior chamber was thoroughly mixed and pipetted in a 96-well plate. Then, its optical density at 492 nm is measured with the microtiter plate photometer.

TREATMENT METHOD: open chamber
In order to apply the test item, the window-locking ring and glass window of the anterior chamber was unscrewed to remove the glass disc. The test item was given on the epithelium that the cornea was evenly covered with test
item. Exposure time of the controls and test item on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red and the final opacity value of each cornea was recorded.

- Corneal opacity: The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
- Corneal permeability: The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
mean of three replicates
Vehicle controls validity:
not applicable
Test item was tested neat.
Negative controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
The test item was tested neat. Under the conditions of this test, the test item Ammonium hexafluorozirconate induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 70.70.
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.
Executive summary:

This in vitro study was performed to assess corneal damage potential of Ammonium hexafluorozirconate by quantitative measurements of changes in opacity and permeability in a bovine cornea.

Six experiments were performed, but two of them were invalid, because one validity criterion of the negative control was not met. These results are not reported but kept with the raw data in the GLP-archive of the test facility. Further three experiments were performed, which led to equivocal results. This is why a sixth experiment was performed with the undiluted test item, because it was assumed that the suspension was not homogeneous. Only this valid experiment is described in detail.


The test item Ammonium hexafluorozirconate 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 neat. Under the conditions of this test, the test item Ammonium hexafluorozirconate induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 70.70.

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.