Bisisopropyl peroxydicarbonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 September 2017 - 21 September 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:

2018

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

Test material information

Constituent 1

Reference substance name:

Bisisopropyl peroxydicarbonate

EC Number:

203-317-4

EC Name:

Bisisopropyl peroxydicarbonate

Cas Number:

105-64-6

Molecular formula:

C8H14O6

IUPAC Name:

2-[({[(propan-2-yloxy)carbonyl]peroxy}carbonyl)oxy]propane

Constituent 2

Reference substance name:

Ethyl acetate

EC Number:

205-500-4

EC Name:

Ethyl acetate

Cas Number:

141-78-6

Molecular formula:

C4H8O2

IUPAC Name:

ethyl acetate

Test material form:

liquid

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, immerged in containers filled with cooled buffered Hanks medium 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)].

Preparation of 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 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL per cornea
- Concentration: undiluted

Duration of treatment / exposure:

Exposure period of 10 minutes (± 30 seconds) at +32°C, followed by rinsing.

Observation period (in vivo):

Not applicable.

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes) at +32°C

Number of animals or in vitro replicates:

Triplicate corneas for each item (test item, negative 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.

TREATMENT
The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 10 minutes (± 30 secondes) in a water bath at +32°C (± 1°C). The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc.) was carried out in the same order for the three corneas of each series.

REMOVAL OF TEST SUBSTANCE
The purpose of rinsing was to eliminate as much item as possible, while taking care not to damage the cornea. On completion of the treatment period, test item, positive and negative controls were removed from the front opening of the anterior chamber (open-chamber method) and epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed three times with pre-warmed cMEM containing phenol red (i.e. until 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.

POST INCUBATION PERIOD
Following the 10-minute treatment and the rinsing step, the holders were incubated for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C). On completio of the 2-hour incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (+32°C (± 1°C)), the second opacity measurement (OPT2) was then performed.

SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
The change in opacity value of each individual cornea treated with test item, negative control or positive control was calculated by subtracting the initial base-line opacity measurement (OPT0) from the post treatment opacity reading (OPT2).
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). When the average change in opacity for the corneas treated with the negative control was negative, it is considered equal to 0.
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 at 4 mg/mL. The holders were incubated with the fluorescein-treated side uppermost) 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 medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).
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 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

Remarks:

test item

Run / experiment:

mean

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

48.0 +/- 17.5

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

MACROSCOPIC EXAMINATION:
No notable opaque spots or irregularities were observed on negative control-treated corneas.
Opacity and fluorescein fixation were observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

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 of the historical mean,
- the mean opacity and mean OD490 nm of the negative control corneas should be less than the established upper limit of historical mean.

In vivo

Irritant / corrosive response data:

No prediction can be made.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

could not be identified as UN GHS Category 1 or as UN GHS No Category.

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation (UN GHS No Category).

Executive summary:

The potential irritant and corrosive properties to the eye of Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test method which 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. 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 negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item was applied undiluted, in a single experiment using a treatment time of 10 minutes and the open-chamber treatment method. Negative and positive controls were applied using the same treatment time but using 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.

Opacity and fluorescein fixation were observed on the three corneas treated with the test item. 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: 16.

As the mean IVIS was > 3 and < 55, the eye hazard potential of the test item could not be predicted. Luperox IPP50EA (50% diisopropyl peroxydicarbonate in ethyl acetate) could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation (UN GHS No Category).