Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
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:
26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: off-white powder
Test item storage: at room temperature

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System:
Bovine eyes were used as soon as possible after slaughter.

Rationale:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
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.

Transport:
Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Amount / concentration applied:
TEST MATERIAL
- 307.7 to 333.9 mg

NEGATIVE CONTROL
- 750 µl of physiological saline per cornea

POSITIVE CONTROL
- 750 µl 20% (w/v) Imidazole per cornea
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
none
Number of animals or in vitro replicates:
3 replicates were used
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

Vehicle
A solubility test in physiological saline was performed. Since no workable suspension of Norethyl in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium Life Technologies).
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader) (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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.
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

Acceptability of the assay
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
-1.1
Negative controls valid:
yes
Remarks:
IVIS -0.9
Positive controls valid:
yes
Remarks:
IVIS 113.4
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The individual in vitro irritancy scores for the negative controls ranged from -2.6 to 0.6. The individual positive control in vitro irritancy scores ranged from 109 to 121. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with Norethyl showed opacity values ranging from -2.8 to -1.0 and permeability values ranging from -0.005 to 0.144. The corneas were clear after the 240 minutes of treatment with Norethyl. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.8 to 0.5 after 240 minutes of treatment with Norethyl.

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 113 and within two standard deviations of the current historical positive control mean (APPENDIX 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

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

Executive summary:

The eye hazard potential of Norethyl was assessed using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD guidelines and GLP principles. The eye damage of Norethyl was tested through topical application for approximately 240 minutes. The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating 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 113 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Norethyl did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 after 240 minutes of treatment. Since Norethyl induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.