[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Cas Number:

2409816-82-4

Molecular formula:

C26H42O4P2

IUPAC Name:

Tributyl(ethyl)phosphonium diphenyl phosphate

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

other: slaughterhouse Müller FleischGmbH, Industriestraße 42, 75217 Birkenfeld, Germany

Details on test animals or tissues and environmental conditions:

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 5 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

10 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 hours at 32 ± 1 °C

Details on study design:

7.2.1 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.
7.2.2 Application
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 of negative control solution, 750 µL oftest item or 750 µL of positive control-were applied to each replicate to the epithelial side of the cornea.
According to the characteristics of the controls, the following treatment procedure was performed:
Closed Chamber Method:
The respective substance (negative control solutionor positive control) 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.
According to the characteristics of the test item, the following treatment procedure was performed:
Open Chamber Method:
In order to apply the test item, the window-locking ring and glass windowof the anterior chamber of each cornea holder was unscrewed to remove the glass disc. The test item was given on the epithelium in a way that the cornea was evenly covered. After dosing, the glass windows are replaced on the anterior chambers to recreate a closed system.
Exposure time of the controls and test item on the corneas was 10 minutes 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 phe-nol red and the cornea holders were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time the final opacity value of each cornea was recorded.
7.2.3 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 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °Cin a hori-zontal 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

Irritation parameter:

in vitro irritation score

Value:

ca. 49.67

Negative controls validity:

valid

Positive controls validity:

valid

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 tributyl(ethyl)phosphonium diphenyl phosphateshowed effects on the cornea of the bovine eye. The calculated mean IVIS was 49.67.

Any other information on results incl. tables

IVIS	UN GHS
≤ 3	No category
> 3 and≤ 55	No prediction can be made
> 55	Eye damage Category I

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study, the test item tributyl(ethyl)phosphonium diphenyl phosphate showed effects on the cornea of the bovine eye. The calculated mean IVIS was 49.67.
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 for eye damage with the BCOP study only. In this case no prediction can be made.