Tetraethylammonium tetrafluoroborate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August - 19 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

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)

Version / remarks:

adopted October 09, 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tetraethylammonium tetrafluoroborate

Cas Number:

429-06-1

Molecular formula:

C8H20BF4N

IUPAC Name:

tetraethylammonium tetrafluoroborate

Test material form:

solid: crystalline

Test animals / tissue source

Species:

cattle

Strain:

other: Bovine

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle
- Characteristics of donor animals: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): test item was weighed and applied directly on the corneas in such a way that the cornea was completely covered (354.1 to 397.9 mg)

Duration of treatment / exposure:

4 hours

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 degrees C. The corneas were incubated for the minimum of 1 hour at 32 degrees C. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

QUALITY CHECK OF THE ISOLATED CORNEAS
Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. 3 corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
A negative control, physiological saline was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
The positive control was a 20% (w/v) Imidazole (CAS No 288-32-4, molecular formula C3H4N2, molecular weight 68.08) solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME
0,75 ml of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Test item was weighed and applied directly on the corneas in such a way that the cornea was completely covered (354.1 to 397.9 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 4 hours at 32 degrees C.

TREATMENT METHOD:
After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

POST-INCUBATION:
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. Corneas were incubated in a horizontal position for 90 minutes at 32 degrees C.

METHODS FOR MEASURED ENDPOINTS:
The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3: No GHS Category
IVIS > 3; ≤ 55: No prediction can be made
IVIS >55: GHS Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 1 (duration of exposure 4 hours)

Value:

4.4 - <= 8.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Tetraethylammonium tetrafluoroborate induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 5.9 after 240 minutes of treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 139 and within two standard deviations of the current historical positive control mean.
- It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since Tetraethylammonium tetrafluoroborate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

Thhe objective of this study was to evaluate the eye hazard potential of Tetraethylammonium tetrafluoroborate as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test item was tested through topical application for approximately 240 minutes. 

The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 5.9 after 4 hours of treatment.

In conclusion, since Tetraethylammonium tetrafluoroborate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.