Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.09.2016
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: bovine
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Freshly isolated bovine cornea
- Characteristics of donor animals (e.g. age, sex, weight): at least 9 month old cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): in HBSS at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects (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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Each isolated cornea was mounted in a cornea holder which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O- ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. 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 position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES: 3

POST-INCUBATION PERIOD: 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 1

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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 spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The test is accepted if:
- The positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- The negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item
Value:
0.31
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The acceptance criteria were met.
Positive control: IVIS = 113.06
Negative control: IVIS = 0.82

Any other information on results incl. tables

Results after 10 Minutes Incubation Time

Test Group

Opacity value = Difference (t130 - t0) of Opacity

Permeability at 490 nm (OD 490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

(IVIS)

Negative Control

0

0.00

0.053

0.054

0.80

0.82

Not categorized

0

0.055

0.83

0

0.055

0.83

Positive Control

112.00*

1.060*

127.90

113.06

Category 1

88.00*

0.987*

102.80

90.00*

1.233*

108.49

Test item

0.00*

0.024*

0.36

0.31

Not categorized

0.00*

0.014*

0.21

0.00*

0.024*

0.36

*corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to the current study and under the experimental conditions reported, the test item is not eye irritant.
Executive summary:

In the current study the corneal damage potential of the test item was assessed by means of the BCOP assay using fresh bovine corneae. The study was according to OECD 437 and GLP.

This test is designed to measure the opacity of the cornea by quantifying the ability of light to pass through it. The permeability, as a result of the irritation potential of the test item, is determined using Na-fluorescein and the opacity before and after the exposure to the test item is compared to determine the damaging effect of the test item. For this purpose the induction of opacity and increased permeability in an isolated bovine cornea after application of the test item is measured and the results of both criteria were combined. The resulting in vitro irritation factor is used for classification as “no category (GHS)”, “no prediction can be made”, and "serious eye damaging" (CLP/EPA/GHS (Cat 1)).

After a first opacity measurement of the fresh bovine corneae, the neat test item, the positive, and the negative controls are applied to the corneae for 10 minutes at 32 ± 1 °C. After the incubation phase the test item, the positive, and the negative controls are washed off and the corneae are further incubated for 120 minutes at 32 ± 1 °C. Afterwards, the opacity is measured a second time.

The opacity measurements are used to determine the permeability of the corneae by measuring spectrophotometrically the transfer of sodium fluorescein after incubation for 90 minutes at 32 ± 1 °C.

The negative control (0.9% (w/v) NaCl solution in deionised water) did not increase the opacity or alter the permeability of the corneae ( mean in vitro irritancy score (IVIS): 0.82) and the positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS: 113.06) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The controls indicated the validity of the test method.

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability and had a calculated in vitro irritancey score (mean IVIS) of 0.31. According to the GHS criteria the test item is not eye irritant as the threshold for serious eye damage is IVIS ≥ 55.

In conclusion, according to the current study and under the experimental conditions reported, the test item is not eye irritant.