Registration Dossier

Diss Factsheets

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:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2017-12-09
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010-10-09
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-Heptanediol
Cas Number:
3710-31-4
Molecular formula:
C7H16O2
IUPAC Name:
1,2-Heptanediol

Test animals / tissue source

Species:
cattle
Strain:
other: 14 month old donor cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Number of animals: Not indicated, but 9 corneae in total were used
- Characteristics of donor animals: 14 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: On the same day the cattle was slaughtered
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects.
- Indication of any antibiotics used: 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) were used in the storage solution and incubation medium
- Selection and preparation of corneas: Only intact corneas were selected, those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
- Quality check of the isolated corneas: Only corneae with a value of the basal opacity < 7 were used.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: The neat test substance was used.
Duration of treatment / exposure:
Ten minutes (± 30 seconds)
Duration of post- treatment incubation (in vitro):
210 minutes (120 minutes until the second second opacity reading, another 90 minutes until permeability measurements)
Number of animals or in vitro replicates:
3 corneas per test item/positive control/negative control
Details on study design:
NUMBER OF REPLICATES: Three replicates

NEGATIVE CONTROL USED: Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED: 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of the neat test item

TREATMENT METHOD: Not indicated

POST-INCUBATION PERIOD: Yes, 210 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium was free of test item, the corneas were given a final rinse with cMEM without phenol red.
- POST-EXPOSURE INCUBATION: In cMEM at 32 ± 1 °C for further two hours in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Using an opacitometer (OP_KiT opacitometer, Electro Design, 63-Riom France). The opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader at OD490

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of three eyes
Value:
64.55
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test item is serious eye damaging (CLP/EPA/GHS (Cat 1).
Executive summary:

This GLP compliant in vitro study according to OECD guideline 437, was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to the different corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured for a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.89).
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 108.29) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
The test item was tested undiluted. Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 64.55 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is classified as serious eye damaging.

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