Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21.06.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.

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. Before the corneas were mounted in corneal holders, they had been visually examined for defects and any defective cornea had been discarded. 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 corneas were incubated for one hour at 32 ± 1 °C

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL of the test substance or the positive respectively negative control substance were introduced into the anterior chamber (closed-chamber method).
Duration of treatment / exposure:
- 4 hours ± 5 minutes incubation at 32 ± 1°C
- Then test and control substances were removed and the epithelium washed at least three times with MEM. 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 illuminance measurement was performed.
- After the illuminance measurement was performed, 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.
Number of animals or in vitro replicates:
- 3 corneas for the test item
- 3 corneas as negative controls treated with physiological saline 0.9% NaCl
- 3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl
Details on study design:
Preparation of the test item
The test item was suspended with physiological saline 0.9% NaCl (AlleMan Pharma, lot no. 609709, expiry date: 21/06/2017) to give a 20% concentration.

Validity of the assay
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean. 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.

Evaluation of results
The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer: Opacity= ( I0/I-b)/a
with a = 0.025 and b = 0.9894
The value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.
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 cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (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 irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)



Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
5.68
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No prediction can be made regarding the classification of the test substance
Other effects / acceptance of results:
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.

Any other information on results incl. tables

Results of the main experiment:

Cornea no. Test item Corrected opacity

Permeability

Corrected OD490 value

IVIS
1 Negative control 0.77 0.021 0.62
2 0.32 0.004
3 0.24 0.01
MV 0.44 0.012
4 Positive control 95.44 3.518 119.95
5 80.82 2.073
6 82.7 1.134
MV 86.32 2.242
7 Test item 4.95 0.031 5.68
8 6.69 0.007
9 4.99 0.002
MV 5.54 0.009

MV = mean value

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The in vitro irritation score was calculated to be 5.78. Hence, no prediction can be made regarding the classification of the test substance.
Executive summary:

The eye irritancy potential of the test substance was investigated in the bovine corneal opacity and permeability assay according to OECD TG 437 and under GLP regulations. The test item was suspended with physiological saline 0.9% NaCl to give a 20% concentration.

All 3 treated corneas showed partial opacity of the tissue. The following mean in vitro irritation score was calculated: 5.68. As a result, no prediction can be made regarding the classification of the test substance according to the evaluation criteria.

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.