(1-methylpropylidene)bis[tert-butyl] peroxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2017 - 04 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 437 (In vitro eye irritation)

Version / remarks:

26 July 2013

Deviations:

yes

Remarks:

The positive control was applied to only 2 corneas instead of 3. Resulting individual and mean IVIS fell within the acceptable range defined in CiToxLAB France historical database and the experiment was consequently considered valid.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(1-methylpropylidene)bis[tert-butyl] peroxide

EC Number:

237-136-7

EC Name:

(1-methylpropylidene)bis[tert-butyl] peroxide

Cas Number:

13653-62-8

Molecular formula:

C14H30O4

IUPAC Name:

2,2'-(butane-2,2-diyldidioxy)bis(2-methylbutane)

Test material form:

liquid

Test animals / tissue source

Species:

cattle

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 immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at CiToxLAB France. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL of the undiluted test item was applied on each cornea.
.

Duration of treatment / exposure:

10 minutes.

Observation period (in vivo):

Opacity measurement:
Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.
The test item, applied undiluted, and the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and 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.
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.

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes)

Number of animals or in vitro replicates:

Triplicate corneas for each tested substance (test item, negative control, positive control).

Details on study design:

EYES SELECTION
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.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

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 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 10 minutes 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 were removed from the front opening of the anterior chamber and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- any test item adhering to the walls of the anterior chamber was removed with a cotton bud,
- the corneas were rinsed five times with pre-warmed cMEM containing phenol red (i.e. until the test item 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 negative control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item 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 (4mg/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 or positive control was calculated by subtracting the average negative control cornea OD490 nm value from the original OD490 nm value of each cornea.
The mean cOD490 nm value of each series of corneas was calculated from the individual cOD490 nm values.

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

ACCEPTANCE OF RESULTS:
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 (SD) of the historical mean,
- the mean opacity and mean OD490 nm of the negative control-treated corneas should be less than the established upper limit of historical mean.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Main

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control.
- Acceptance criteria met for positive control.

In vivo

Irritant / corrosive response data:

Not requiring classification for eye irritation or serious damage (UN GHS No Category).

Other effects:

OTHER EFFECTS (macroscopic examination):
Negative control and test item-treated corneas: None
Positive control-treated corneas: Opacity, fluorescein fixation and thickening of each cornea

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 not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The potential irritant and corrosive properties of the LUPEROX® 520M50 to the eye was evaluated with the Bovine Corneal Opacity and Permeability (BCOP) test method. The BCOP assay 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 procedures and 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 experiment was performed using three corneas for test item and negative control and two corneas for positive control. Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, applied undiluted, and the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and 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. 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 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 asa testchemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Under the experimental conditions of this study, LUPEROX® 520M50 was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).