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EC number: 248-363-6
CAS number: 27247-96-7
Skin irritation / corrosion: not irritating upon one contact, but a
repeat-dose DNEL can be set from the data of the 3-week dermal study in
rabbits. For this purpose, a repeat-dose local NOAEC was derived as 0.22
Eye irritation: not irritating
In the experimental conditions of the study, the test item was
considered to be not irritating to the skin.
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.
The test item was first administered to a single male New Zealand
White rabbit. Since the test item was not severely irritant on this
first animal, it was then evaluated simultaneously in two other animals.
A single dose of 0.1 mL of the undiluted test item was instilled
into the left conjunctival sac. The right eye was not treated and served
The eyes were not rinsed after administration of the test item.
Ocular reactions were observed approximately 1 hour, 24, 48 and
72 hours after the administration.
The mean values of the scores for chemosis, redness of the
conjunctiva, iris lesions and corneal opacity were calculated for each
The interpretation of results was carried out according to the
classification criteria laid down in Council Directive 67/548/EEC (and
A slight chemosis was noted in all animals on day 1. Slight or
moderate redness of the conjunctiva was observed in all animals on day
1; persisting until day 2 in two of them. A clear discharge was recorded
on day 2 in 1/3 animals.
Mean scores calculated for each animal over 24, 48 and 72 hours
were 0.0, 0.0 and 0.0 for chemosis, 0.0, 0.3 and 0.3 for redness of the
conjunctiva, 0.0, 0.0 and 0.0 for iris lesions and 0.0, 0.0 and 0.0 for
Under the experimental conditions of the study, the test item
2-Ethylhexyl nitrate was slightly irritant when administered by ocular
route to rabbits.
However, according to the classification criteria laid down in
Council Directive 67/548/EEC (and subsequent adaptations), the test item
should not be classified as irritating to the eyes.
The following concentration-response data are available for single
application in rabbits:
- study N° EB3413: highly irritant at an excessive concentration (vs
recommendation) of 0.5 mL over 2.5 cm2 skin (too small surface) over 4h
= 192 mg/cm2;
- study N° 3552 TAL: non irritant at the recommended level
of 0.5 mL over 6 cm2 skin over 4h = 0.962 * 0.5 / 6 g/cm2 = 80 mg/cm2;
a minimal erythema started at 48 or 72h and lasted for <1 week;
- study N° 80-2189A, on day 1: non-irritant (at most: one grade 2 edema
out of 3 animals, no other reaction) at 7.7 mg/cm2 (high-dose), applied
over 6h to non-abraded animals.
These data show that single application leads to concentration-dependent
results. The relevant conclusion is that of the test performed at the
recommended concentration. The importance and persistence of reactions
in the first (excessive concentration) study may also suggest an impact
The substance induced delayed atypical skin reactions (there is
no classification for such properties) as suggested by the standard
assay above, and largely confirmed by marked irritation and skin
cracking after repeated applications to rabbits (the same animals showed
no relevant effect within the first three applications). As indicated in
the sensitisation section (7.4), this was not attributable to
sensitisation in the absence of histological signs of immunological
reaction in Magnusson and Kligman assays. It seems possible that this
effect partly corresponds to vasodilation (2-ethylhexyl nitrate
being an organic nitrate so possibly a nitric oxide donor). However,
skin cracking was also observed in the repeated application study in
rabbits, and could also suggest interaction with skin lipids due
to the very high log Kow value. Last, an impact of impurities
could be suspected: the IUCLID4 file (see attached file) of the
synthesis precursor 2EH, which may be present as an impurity in some
studies (no data), is attached and shows that this substance is clearly
a skin irritant.
Minimally irritant (this is the minimal possible conclusion grade) in
vitro (BCOP). Non irritant in vivo in rabbits.
It is noteworthy that the IUCLID4 file of the synthesis precursor 2EH
(see attached file), which may be present as an impurity in some studies
(no data), shows that this substance induces some eye irritation but
apparently not of a level requiring classification.
No data. Based on absence of irritation for skin and eyes, no irritation
to respiratory tract is expected.
Non irritant to skin and eyes based on standard
single-application studies in rabbits.
NB: for the skin, this conclusion is only valid for a
sufficiently pure substance due to a possible impact of impurities (e.g.
EUH 066 and R66 are
applicable due to skin cracking and dryness after repeated dermal
No conclusion for respiratory tract.
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