Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 December 2015 -- 04 February 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 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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyl 4,4-bis(tert-butyldioxy)valerate
EC Number:
213-626-6
EC Name:
Butyl 4,4-bis(tert-butyldioxy)valerate
Cas Number:
995-33-5
Molecular formula:
C17H34O6
IUPAC Name:
butyl 4,4-bis(tert-butylperoxy)pentanoate
Test material form:
other: Colorless to yellow liquid

Test animals / tissue source

Species:
cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: bovine cattle were up to 12 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 swivelled 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 within a maximum of 24 hours.

(Pre)Incubation T°C: 32°C
Dates of experimental phase: from 08 January 2016 to 04 February 2016

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Amount applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
Exposure period of 10 minutes (± 30 seconds) followed by rinsing
Observation period (in vivo):
Opacity measurement:
- before treatment
- after 2-hour incubation in water bath at +32°C (± 1°C).

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement
Number of animals or in vitro replicates:
Not applicable
Triplicate corneas for each timepoint and tested substance (test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Rinsing: - the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed six times with pre-warmed cMEM containing phenol red (i.e. until the dose 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.

NEGATIVE CONTROL:
As the test item was tested undiluted (i.e. in its original form), 0.9% Sodium Chloride (0.9% NaCl) was used as negative control.

Since several test items were assayed concurrently, the negative control was shared.

POSITIVE CONTROL:
As the test item was tested using a 10-minute treatment, the positive control was absolute ethanol. It was used neat and sampled on the day of use.

Since several test items were assayed concurrently, the positive control was shared.

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values of positive control and test item.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values of positive control and test item.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
39
Irritation parameter:
cornea opacity score
Value:
0.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
10
Irritation parameter:
fluorescein leakage
Value:
0.006
Vehicle controls validity:
valid
Remarks:
0.005
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
1.911

Applicant's summary and conclusion

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

The potential irritant and corrosive properties of Luperox 230 the eye was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test. This 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 guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice. 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 validated experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, tested undiluted, and the negative and positive controls were applied in a single validated experiment using a treatment time of 10 minutes and using the closed-chamber. At completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was 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. No notable opaque spots or irregularities were observed on all test item-treated corneas. 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). Under the experimental conditions of this study, Luperox 230 was identified as a test chemical not requiring classification for eye irritation.