Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-11-08 to 2016-11-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented GLP-study according to OECD Guideline 437.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

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 material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): JNJ-42398681-AAA (T001589)
- Physical state: liquid
- Appearence: light brown
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16FB3011
- Expiration date of the lot/batch: 2018-03-14 (retest date)
- Purity: 87.4%
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, the test item was applied undiluted

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the test item was applied undiluted





Test animals / tissue source

Species:
other: Freshly isolated bovine cornea
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Vitelco, -'s Hertogenbosch, The Netherlands
- Bovine eyes were used as soon as possible but within 4 hours after slaughter. Bovine eyes from young cattle were obtained from the slaughterhouse, 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 and tested the day of arrival in the laboratory


Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): >/= 99.9%


Duration of treatment / exposure:
Corneas were incubated for 10 ± 1 minutes at 32 ± 1°C
Duration of post- treatment incubation (in vitro):
After 10 ± 1 minutes of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed after the incubation period of 90 minutes ± 5 minutes following the opacity measurement.
Number of animals or in vitro replicates:
3 corneas were selected at random for each treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- 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.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal 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°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.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µl of the negative control, Ethanol (positive control) or test item were introduced onto the epithelium of the cornea. The holder was 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 10 ± 1 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed with MEM with phenol red (Eagle’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.

METHODS FOR MEASURED ENDPOINTS
-CORNEAL OPACITY: Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be recorded. Corneas that had an initial opacity reading higher than 7 were not used.
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (l = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to:
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 before/after test item treatment. The change of opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item 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.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader)
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 4 mg Nafluor escein/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 e ach 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 ra nge (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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to c alculate 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.

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of IVIS score of test item: 0.2 to 0.8
Irritation parameter:
cornea opacity score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of corneal opacity score of test item: 0.2 to 0.9
Irritation parameter:
other: permeability score
Remarks:
mean of 3 eyes
Run / experiment:
1
Value:
0.004
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: range of permeability value of test item -0.007 to 0.015
Other effects / acceptance of results:
mean in vitro irritancy score (range):
negative control: -0.2 (-1.1 to 3.4*) *Excluded from analysis (study plan deviation 1).
positive control: 50.2 (43.6 to 56.3)

mean opacity scores (range):
negative control: -0.4 (-1.4 to 3.1** ) ** Excluded from analysis (study plan deviation 1)
positive control: 19.0 (14.8 to 24.5)

mean permeability scores (range):
negative control: 0.013 (0.003 to 0.023)
positive control: 2.082 (1.917 to 2.213 )

The corneas treated with the test item showed opacity values ranging from 0.2 to 0.9 and permeability values ranging from -0.007 to 0.015. The corneas were slightly translucent after the 10 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 0.2 to 0.8 after 10 minutes of treatment with the test item.

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. One of the negative control eyes was excluded from the analysis, since the opacity value of 3.1 was just ouside the historical control data range of -2.9 – 3.0 resulting in an IVIS score >3 (Study Plan Deviation 1). The mean in vitro irritancy score of the positive control (Ethanol) was 50.2 (43.6 to 56.3) and was within two standard deviations of the current historical positive control mean. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Final Report Amendment No. 1 was issued on 2018-04 -25. The amendment was issued at the request of the sponsor and the new Certificate of Analysis and Technical Document had no impact on the interpretation of the study results.

Final Report Amendment No. 2 was issued on 2018 -09 -21. The amendment was issued at the request of the sponsor and the new Technical Document has no impact on the interpretation of the study results.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.