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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:
2015-2016
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
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 26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 040802#
- Expiration date of the lot/batch: 06.08.2016
- Purity test date: 08/01/2015
- Purity: 99.17%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel:suspended with physiological saline 0.9% NaCl to gain a 20% concentration


FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Remarks:
beef cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):fresh eyes collected from the slaughterhouse on test day and transported in HBSS containing Pen/Strep on ice to the laboratories. Corneas are then incubated with RPMI (1% Fetal Bovine Serum and 2 mM L-glutamine, without phenol red)) for 1 hour at 32 +/-1 °C

- Time interval prior to initiating testing: 1 hour
- indication of any existing defects or lesions in ocular tissue samples:corneas were visually carefully examined for defects prior equilibrium storage and any defective cornea had been discarded also after a first check of opacity after equilibrium.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 µl
- Concentration (if solution): 20% in NaCl 0.9%

VEHICLE / NEGATIVE CONTROL : physiological saline NaCl 0.9%
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 1406788/B Braun

POSITIVE CONTROL
- Amount apply: 750 µl
- Name: imidazole 20% in physiological saline 0.9%
- Lot/batch no. : SLBK9670V : Sigma



Duration of treatment / exposure:
4 hours +/- 5 minutes
Duration of post- treatment incubation (in vitro):
90 min at 32 +/-1°C
Number of animals or in vitro replicates:
3 per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera.
The isolated corneas were stored in a petri dish containing HBSS. After quality check step 1, the isolated corneas were mounted in corneal holders (BASF, Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32  1 °C.
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer (Quality check step 2).

QUALITY CHECK OF THE ISOLATED CORNEAS
Step 1: The cornea had been visually examined for defects and any defective cornea had been discarded.
Step 2: Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay

NUMBER OF REPLICATES 3 per group

NEGATIVE CONTROL USED
3 corneas treated with physiological saline 0.9% NaCl

POSITIVE CONTROL USED
3 corneas treated with imidazole 20% in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME: 750 µl during 4 hours +/- 5 minutes at 32 +/- 1 °C
TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
ca. 1.41
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: (opacity and permeability values < to the established upper limits for background bovine corneas) YES
- Acceptance criteria met for positive control:(within the 2 stndard deviations of the current historical mean, within 97.2 and 161.84 in vitro irritation score)
- Range of historical values if different from the ones specified in the test guideline:within 97.2 and 161.84 in vitro irritation score

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The eye irritancy potential of X330 was investigated in the bovine corneal opacity and permeability assay.
The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration.
The following mean in vitro irritation score was calculated: 1.41
Therefore the test item was classified into UN GHS No Category.
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Executive summary:

The eye irritancy potential of the test substance was investigated in the bovine corneal opacity and permeability assay according to the OECD guideline number 437. The test item was suspended with physiological saline 0.9% NaCl to gain a 20% concentration. The following mean in vitro irritation score was calculated: 1.41. Therefore the test item was classified into UN GHS No Category. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

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