Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

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

Test material

Constituent 1
Test material form:
solid: crystalline

Test animals / tissue source

Species:
other: bovine corneal opacity and permeability assay

Test system

Vehicle:
physiological saline
Controls:
other: 3 corneas as negative controls treated with physiol. saline 0.9 % NaCl and 3 corneas as positive control treated with imidazole 20 % in physiological saline 0.9 % NaCl
Amount / concentration applied:
20 % concentration of the test substance in physiological saline 0.9 % NaCl, applied on 3 corneas
Duration of treatment / exposure:
750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours +/- 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed.
Number of animals or in vitro replicates:
Not applicable, in vitro test / 3 replicates
Details on study design:
Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI.
An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France).
Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as
negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that
have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and
replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium
washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed
with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was
refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and
the corneas were incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed
and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.


Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading.
These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas.
The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490).
Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500),
were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article
and the positive control were calculated by subtracting the average corrected OD490 of the negative control corneas from
the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated
corneas for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3
Value:
82.55
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The eye irritancy potential of the substance was investigated in the bovine corneal opacity and permeability assay. The substance was diluted with physiological saline 0.9 % NaCl to gain a 20 % concentration.

The following mean in vitro irritation score was calculated: 82.55

Therefore the substance was classified as very severe irritant.

Applicant's summary and conclusion

Interpretation of results:
other: very severe irritant
Remarks:
Criteria used for interpretation of results: other: OECD 437
Conclusions:
As a result of the BCOP test, the substance is classified as very severe eye irritant.
Executive summary:

The eye irritancy potential of the substance was investigated in the bovine corneal opacity and permeability assay (in vitro test). The mean in vitro irritation score of the substance was determined to be 82.55. According to the evaluation criteria the substance is classified as very severe eye irritant.