Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June 2017 - 24 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Nitrosodiphenylamine
EC Number:
201-663-0
EC Name:
Nitrosodiphenylamine
Cas Number:
86-30-6
Molecular formula:
C12H10N2O
IUPAC Name:
N-nitroso-N-phenylaniline
Test material form:
solid: pellets

Test animals / tissue source

Species:
cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Species: bovine cattle (Bos Taurus).
- Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
- Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
- Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.


Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

- Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
- Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
- Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used immediately.

Test system

Vehicle:
other: paraffin oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied = 750 µL
- Concentration = 20% (w/v) in vehicle
Duration of treatment / exposure:
Exposure period of 4 hours (± 5 minutes), followed by rinsing
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
Triplicate corneas for each tested substance (test item formulation, vehicle control, positive control)
Details on study design:
EYES SELECTION
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number. After pre-incubation, corneas that showed any macroscopic defect or an opacity value over 7 were discarded.

TREATMENT
The three corneas from the same series were always processed in the same order at each step.
The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 4 hours in a water bath at +32°C (± 1°C).

REMOVAL OF TEST SUBSTANCE
The purpose of rinsing was to eliminate as much items as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item formulation was removed from the front opening of the anterior chamber and the epithelium was rinsed as follows:
- the residual test item formulation adhering to the walls of the anterior chamber was first removed with a cotton bud and then using a pipette of heated cMEM (32°C),
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item formulation had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. The average change in opacity for the corneas treated with the vehicle control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item formulation or positive control to obtain a corrected opacity value (cOPT). The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

- Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution (5mg/mL) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube.
The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item formulation or positive control was calculated by subtracting the average vehicle control cornea OD490 nm value from the original OD490 nm value of each cornea. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.

- Scoring:
In vitro irritancy score (IVIS) = cOPT + (15 x cOD490 nm)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
MACROSCOPIC EXAMINATION:
Fluorescein fixation was observed on all corneas treated with the vehicle control and the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.


ACCEPTANCE CRITERIA:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
- the mean opacity and mean OD490 nm of the vehicle control corneas should be less than the established upper limit of historical mean.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
not eye irritating
Conclusions:
Under the experimental conditions of this study, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).
Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice.

 

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and vehicle control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was tested at 20% (w/v) in paraffin oil, in a single experiment using a treatment time of 4 hours and the open-chamber treatment method. Vehicle and positive controls were applied using the same treatment time but the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

A second opacity measurement was then performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes) at +32°C.

At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

 

Results

 

Macroscopic examination

Fluorescein fixation was observed on the three corneas treated with the test item.

 

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

 The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

 As the mean IVIS was < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Conclusion

Under the experimental conditions of this study, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).