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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2020 - 13 October 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
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:
June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione
Cas Number:
54024-17-8
Molecular formula:
C20 H26 O2
IUPAC Name:
(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione
Test material form:
solid: particulate/powder
Details on test material:
Storage Conditions: In refrigerator (2-8°C)
Specific details on test material used for the study:
Physical Description: Off-white powder
Storage conditions: In refrigerator (2-8°C)
Test item handling: No specific handling conditions required

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as
soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
- Time interval prior to initiating testing:
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Selection and preparation of corneas: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were counted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 309.66 to 369.88 mg

NEGATIVE CONTROL
- Amount applied: 750 µL

POSITIVE CONTOL:
- Amount applied: 750 µL
- Concentration 20% (w/v)
Duration of treatment / exposure:
240 ± 10 minutes at 32 ± 1°C.
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes at 32 ± 1°C.
Number of animals or in vitro replicates:
Three corneas were selected.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with MEM with phenol red

- POST-EXPOSURE INCUBATION: The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: ((𝐼0/𝐼)− 0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
>= -2.7 - <= -0.7
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
CONTROLS
The individual in vitro irritancy scores for the negative controls ranged from 1.4 to 5.0. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 122 to 163. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

ACCEPTANCE OF RESULTS
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 142 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

RESULTS
The corneas treated with the test item showed opacity values ranging from -3.1 to -1.0 and permeability values ranging from 0.017 to 0.025. The corneas were clear/translucent after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.7 to -0.7 after 240 minutes of treatment with the test item.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.9 after 240 minutes of treatment.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
A BCOP test was performed according to OECD TG 437 and in accordance with GLP principles. In conclusion, since EMETAM induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

A BCOP test was performed according to OECD TG 437 and in accordance with GLP principles. The objective of this study was to evaluate the eye hazard potential of EMETAM as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). 


Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 142 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system
functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.9 after 4 hours of treatment.
In conclusion, since EMETAM induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.