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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-12-11
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
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:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-bis(methoxymethyl)benzene
EC Number:
229-828-2
EC Name:
1,4-bis(methoxymethyl)benzene
Cas Number:
6770-38-3
Molecular formula:
C10H14O2
IUPAC Name:
1,4-bis(methoxymethyl)benzene
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: A7503
Purity: 99.89%

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands),
- Characteristics of donor animals: young cattle
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: No

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): 750 μL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Preparation of Corneas: 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) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
- Cornea Selection and Opacity Reading: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. 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.
QUALITY CHECK OF THE ISOLATED 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.
NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.
NEGATIVE CONTROL USED : physiological saline
POSITIVE CONTROL USED : Ethanol
APPLICATION DOSE AND EXPOSURE TIME
750 μL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: 120 ± 10 minutes at 32 ± 1°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea. The light was measured as illuminance by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: Possible pH effects of the test item on the corneas were recorded

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
5.4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: mean of 3 treatment
Other effects / acceptance of results:
OTHER EFFECTS:
The corneas treated with test item showed opacity values ranging from 3.4 to 7.5 and permeability values ranging from 0.006 to 0.068. The corneas were translucent (with a clear rim) after the 10 minutes of treatment with test item. No pH effect of the test item was observed on the rinsing medium.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made
Conclusions:
In conclusion, since the test item induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of test item 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) based on OECD 437.

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of test item was tested through topical application for 10 minutes.

The test item was applied as it is (750 μL) directly on top of the corneas. All test validation criteria met, it was therefore concluded that the test conditions were adequate and that the test system functioned properly. The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 5.4 after 10 minutes of treatment.

In conclusion, since test item induced an IVIS >3 <=55, no prediction on the classification can be made.