Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 19 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
other: in-vitro test
Strain:
other: isolated bovine cornea
Details on test animals or tissues and environmental conditions:
TEST MATERIAL
- Source: slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Acclimation period: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. Then, the corneas were dissected and incubated in incubation medium at 32 +/- 1°C in an incubation chamber for 1 hour.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 +/- 1°C

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
An amount of 750 µl of either the negative control, positive control or test item was applied directly on the corneas in such a way that the cornea was completely covered.
Duration of treatment / exposure:
The corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1°C.
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes at 32+/-1°C.
Details on study design:
The aim of this study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea.

PROTOCOL
- For each treatment group (negative control, positive control and test item), three replicates were used.
- The opacity of each cornea was determined before exposure.
- The corneas were exposed for 10 minutes.
- After the incubation the change in opacity for each individual cornea (including the negative control) was determined.
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.

INTERPRETATION
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro irritancy score (IVIS)
- A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritant.
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
59
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test substance showed opacity values ranging from 30 to 34 and permeability values ranging from 1.482 to 2.287. The corneas were turbid after the 10 minutes of treatment with Tributyl(ethyl) phosphonium diethylphosphate. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, the in vitro irritancy scores ranged from 56 to 64 after 10 minutes of treatment with Tributyl(ethyl) phosphonium diethylphosphate.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean 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 (Ethanol) was 47 and was within two standard deviations of the current historical positive control mean.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Tributyl(ethyl) phosphonium diethylphosphate induced serious eye damage in the Bovine Corneal Opacity and Permeability test.
Executive summary:

A GLP compliant in-vitro study was performed according to OECD Guideline 437 in order to evaluate the ocular irritation properties of Tributyl(ethyl) phosphonium diethylphosphate on an isolated bovine cornea. The possible ocular irritancy of the test item was tested through topical application for approximately 10 minutes. For each treatment group (negative control, positive control and test item), three replicates were used. After removal of the test item and two hours post-incubation, opacity and permeability values were measured. After treatment, the IVIS (in vitro irritancy score) in the controls is within the acceptability criterion. The positive control showed clear irritating effects. Tributyl(ethyl) phosphonium diethylphosphate induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 59 after 10 minutes of treatment.