Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Dec 2018 - 18 Dec 2018
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 guidelineopen allclose all
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:
2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide
Cas Number:
943986-70-7
Molecular formula:
C11H20N2O2
IUPAC Name:
Reaction mass of (S)-2-[(R)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide and (S)-2-[(S)-2-Oxo-4-propyl-1-pyrrolidinyl]butyramide
Test material form:
solid
Details on test material:
- Appearance: Off-white solid
- Storage condition of test material: At room temperature protected from light

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL: 750 µL of a 20% (w/v) solution in physiological saline per cornea
POSITIVE CONTROL: 750 µL of a 20% (w/v) Imidazole solution in physiological saline per cornea
NEGATIVE CONTROL: 750 µL of physiological saline per cornea
Duration of treatment / exposure:
4 hours (at 32°C)
Duration of post- treatment incubation (in vitro):
Not applicable
Number of animals or in vitro replicates:
3 corneas for each treatment group
Details on study design:
SELECTION, PREPARATION AND QUALITY CHECK OF CORNEAS
- 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. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- 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. Corneas with those exhibiting defects were discarded.
- The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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°. 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, BASF, Ludwigshafen, Germany). 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.

TREATMENT
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

OPACITY
The opacity of a cornea was measured by the diminution of light passing through the cornea. The change in opacity for each individual cornea was calculated (also for the negative control), by subtracting the initial opacity reading from the final post-treament reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

PERMEABILITY
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. 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 (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
- The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

DECISION CRITERIA
- A test item that induces an IVIS >55 is classified with Eye Irr. Cat. 1 in accordance with the CLP Regulation (and GHS)
- A test item that induces an IVIS ≤ 3 is not classified for Eye irritation/corrosion in accordance with the CLP Regulation (and GHS)
- For a test item that induces an IVIS of >3 - ≤55, no prediction on the classification can be made.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of 3 replicates
Value:
367
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- The individual in vitro irritancy scores for the negative controls ranged from -0.5 to 1.6. The corneas treated with the negative control item were clear after the 240 minutes of treatment.
- The corneas treated with the test item showed opacity values ranging from 152 to 653 and permeability values ranging from 1.774 to 2.740. The corneas were turbid after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. The individual in vitro irritancy scores were 232, 178 and 691 for the corneas treated with the test item.
- The individual in vitro irritancy scores of the positive control were 149, 118 and 147. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (opacity: -0.5 - 1.4; permeability: 0.000 - 0.008)
- Acceptance criteria met for positive control: Yes (IVIS: 138)
- Historical Control Data (period: Nov 2016 to Nov 2018):
Negative control Positive control
Opacity Permeability In vitro Irritancy Score In vitro Irritancy Score
Range -4.4 – 5.2 -0.011 - 0.081 -4.3 – 5.4 93 – 206
Mean 1.12 0.02 1.13 146.67
SD 1.92 0.02 1.96 26.95
n 112 112 111 112

Any other information on results incl. tables

Summary of results

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score

Negative control

0.5

0.004

0.5

Positive control

120

1.225

138

Test item

332

2.337

367

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (irreversible effects on the eye) based on GHS and CLP criteria
Conclusions:
Based on an in vitro irritancy score of 367 in a Bovine Corneal Opacity and Permeability test, it is concluded that the substance is classified Category 1 (irreversible effects on the eye) for eye irritation/corrosion according to Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

Using the Bovine Corneal Opacity and Permeability test (BCOP test) the substance was screened for its eye hazard potential in accordance with OECD 437 and according to GLP principles. The solid test item was applied as a 20% (w/v) solution in physiological saline. Adequate negative and positive controls were included. The substance induced serious eye damage, resulting in a mean in vitro irritancy score of 367 after 4 hours of treatment. The acceptability criteria were met. Since the substance induced a mean IVIS > 55, in all 3 corneas, the substance is classified Category 1 (irreversible effects on the eye) for eye irritation/corrosion according to Regulation (EC) No. 1272/2008 (CLP).