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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:
key study
Study period:
07 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
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 Jul 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

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

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, Brensbach, Germany
- Characteristics of donor animals: 19 - 59 months old
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes and the eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 20% (w/v) in 0.9% sodium chloride solution

VEHICLE
- Amount(s) applied: 750 µL

POSITIVE CONTROL
- Amount(s) applied: 750 µL
- Concentration: 20% (w/v) in 0.9% sodium chloride solution
Duration of treatment / exposure:
240 min at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 min at 32 ± 1 °C
Number of animals or in vitro replicates:
Triplicates for each treatment and control group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a specially designed cornea holder.

QUALITY CHECK OF THE ISOLATED CORNEAS
The baseline opacity was determined with a calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). Any corneas that showed macroscopic tissue damage (scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES
Triplicates for each treatment and control group

TREATMENT METHOD
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (0-ring) of the posterior part of the holder. The cornea was gently flattened over the 0-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form. For equilibration, the corneas in the holder were incubated (BSS 160, Grumbach Brutgeräte GmbH, Asslar, Germany) in a vertical position at 32 ± 1 °C for about one hour.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurements.
- POST-EXPOSURE INCUBATION: After the rinsing step, the corneas were incubated again in an incubator in a horizontal position at 32 ± 1 °C for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The light transmission through the corneas, given as lux value, was recorded and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: The amount of fluorescein that crossed the cornea was measured spectrophotometrically (OD490) in the medium from the posterior chamber.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as Category 1.
Test substance with an IVIS ≤ 3 was regarded as No Category.
Test substance with an IVIS > 3 ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean value of 3 corneae
Value:
1.4
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
No observations (tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.0 and, therefore within three standard deviations of the current historical mean of the negative control (IVIS: -1.5 to 3.5).
- Acceptance criteria met for positive control: After treatment with the positive control (20% imidazole) the calculated IVIS was 107.7 and therefore falls within two standard deviations of the current historical mean of the positive control (IVIS 78.2 to 135.3).

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
Under the conditions of the BCOP assay, the test substance did not show irritant properties towards eyes.
CLP: not classified