Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th april 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This study should provide a rational basis for risk assessment in humans. An ocular contact is one of the possible routes of human exposure.
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 solution. The comparison of the opacity before and after the exposure to the test item and the determination of the permeability after the treatment provide an indication of 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 was measured. The results of both criteria were combined. The resulting in vitro irritation factor was compared with the classification “no Category (GHS)”, “no prediction can be made”, and "serious eye damaging" (CLP/EPA/GHS (Cat 1)).
The purpose of this study was to assess the possible corneal damage potential when fresh bovine corneae are exposed to the test item.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
White powder
Specific details on test material used for the study:
Batch: 18114773
Expiry Date: October 2018

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as 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. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, 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 (Oring) 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.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was tested as a 20% suspension (w/v) in saline using ultrasonic technique for 10 minutes.
The anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
Duration of treatment / exposure:
The incubation time lasted 240 minutes
Number of animals or in vitro replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
240 min
Value:
> 1 - < 3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: permeability
Run / experiment:
490 min
Value:
>= 0.004 - <= 0.019
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported,
1574 ACIDE CRIST is not categorized (GHS).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of 1574 ACIDE CRIST by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item 1574 ACIDE CRIST, the positive and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item 1574 ACIDE CRIST did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 1.08.

According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (GHS).