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EC number: 248-363-6
CAS number: 27247-96-7
For each experiment, the acceptancecriteria were fulfilled:
individual corneal opacity values of negative controls were < 10 in
individual OD490 nmvalues of negative control corneas were <
0.100 in both experiments,
solution of fluoresceine (at 5 mg/mL in DPBS) diluted 1:1000 in cMEM had
OD490 nm values between 0.850 and 0.940
inb oth experiments,
the 30-minute treatment, the positive control mean in vitro scores
was 124.1, thus demonstrating the sensitivity of the test system under
the experimental conditions of this study.
The corneas were obtained from the eyes of freshly slaughtered
calves at the abattoir. They were mounted in the corneal holders with
the endothelial side against the O-ring of the posterior half of the
holder. Both compartments of the corneal holder were filled in excess
with Minimal Essential Medium Eagle completed with 1% fetal calf serum
plus penicillin/streptomycin (cMEM), then the holders were preincubated
for 1 hour at 32°C.
Three corneas were used for each treated series (test item,
positive control and negative control).
Before the treatment, a first opacity measurement was performed
using an opacitometer (determining the light transmission through the
center of each mounted cornea).
For the treatment, the test item was used undiluted.
The test item was tested sequentially in two consecutive
As the mean in vitro score at the 30-minute treatment was ≤
10, the second experiment was undertaken using a 4-hour treatment.
At the completion of the treatment period, the test item was
removed from the front opening of the anterior part of the holder and
the epithelium was washed.
Following the 30-minute treatment, the corneas were incubated for
2 hours at 32°C. At the completion of the 2-hour incubation period, the
second opacity measurement was performed.
Following the 4-hour treatment, the second opacity measurement was
performed immediately without any further incubation after the rinsing
of the dosage form.
After the second opacity measurement, the medium was removed from
both compartments of each holder. The posterior compartment was refilled
with cMEM at, while the anterior compartment received 1 mL of a 5 mg/mL
fluoresceine solution in Dulbecco's Phosphate-Buffered Saline (DPBS).
Then, the holders were incubated vertically for 90 minutes at 32°C.
At the end of the 90-minute incubation, the optical density of the
solution from the posterior compartment of the holder was measured at
490 nm in order to determine the permeability of the cornea. Then the
cornea was removed from the holder and observed for opaque spots and
For each experiment, the acceptance criteria were fulfilled and
the study was therefore considered to be valid.
No notable opaque spots or irregularities were observed on
negative control corneas, either following the 30-minute treatment or
following the 4‑hour treatment.
No notable opaque spots or irregularities were observed on test
item-treated corneas, following the 30-minute treatment while
fluoresceine fixation was noted on corneas, following the
Following the 30-minute treatment, the mean in vitro score
was 3.0. Then following the 4‑hour treatment, the mean in
vitro score was 6.2.
Under the experimental conditions of this study, according to both
mean in vitro scores of the 30‑minute and 4-hour treatments, the
test item 2-Ethylhexyl nitrate tested in its original form is classified
as slightly irritant for the isolated calf cornea.
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