Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

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:
09 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the United Kingdom, Medicines and Healthcare Products, Regulatory Agency, United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: obtained from a local abattoir as a by-product from freshly slaughtered animals
- Number of animals: not reported
- Characteristics of donor animals: adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue: The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3 corneas for the test item
3 corneas as negative controls treated with 0.9% NaCl
3 corneas as positive controls treated with ethanol
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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 iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle's Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
yes; 0.75 mL sodium chloride 0.9% w/v

POSITIVE CONTROL USED
yes; 0.75 mL ethanol

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL test material for 10 minutes

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: test substance or the control substance was removed and the epithelium washed at least three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.
- POST-EXPOSURE INCUBATION: 120 minute incubation at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.
- Corneal permeability: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

SCORING SYSTEM: The following formula was used to determine the in vitro irritation score (IVIS): IVIS = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA: The BCOP assay was 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 collated during the previous 12 months for this testing facility. For the negative control, BCOP assay was considered to be valid if the in vitro irritation score obtained was less than or equal to the upper limit for background opacity and permeability values calculated from the previous 12 months data for this testing facility.

The following criteria was used for the BCOP Assay: ≤ 3 = not inducing eye damage; > 3; ≤ 55: no prediction can be made; and > 55: inducing serious eye damage. When no prediction for inducing serious eye damage or not inducing eye damage can be made further testing with another suitable method is required.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
0.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes; The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: yes; The positive control in vitro irritancy score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification required according to Regulations (EC) No 1272/2008.
Conclusions:
Under the conditions of the test, the test substance was shown to have no irritation or corrosive potential in the bovine corneal opacity and permeability (BCOP) test prediction model. The mean in vitro irritation score was 0.9.