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EC number: 701-362-9
CAS number: -
No unscheduled deaths and no clinical signs were observed during the
No local reactions were observed in any animals. No notable increase in
ear thickness was observed at any tested concentrations.
No notable lymphoproliferation was noted with the test item at any
The objective of this study was to
evaluate the potential of the test item to induce delayed contact
hypersensitivity using the murine Local Lymph Node Assay (LLNA).
This study was conducted in compliance
with the principles of Good Laboratory Practice.
To assess the irritant potential of
the test item (through ear thickness measurement), a preliminary test
was first performed in order to define the test item concentrations to
be used in the main test. Two groups of two female mice received the
test item by topical route to the dorsal surface of both ears (one
concentration per ear) on days 1, 2 and 3 at concentrations of 10, 25,
50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day
6, the thicknessof both ears of each animal was measured and the local
reactions were recorded. Each animal was observed at least once a day
for mortality and clinical signs. Body weight was recordedonce during
the acclimation period, and thenon days 1 and 6. On completion of the
observation period, the animals were sacrificedthen discarded without
macroscopic post-mortem examination.
In the main test, five groups of four
female mice received the test item by topical route to the dorsal
surface of both earson days 1, 2 and 3 at concentrations of 5, 10, 25,
50 or 100% under a dose-volume of 25 µL. One negative control group of
four females received the vehicle (acetone/olive oil (4/1;
v/v)) under the same experimental conditions. Additionally,
one positive control group of four females received the
positive control, a-hexylcinnamaldehyde (HCA), at 25% in
a mixture acetone/olive oil (4/1; v/v) under the same
From day 1 to day 3, then on day 6,
the thickness of the left ear of each animal was measured, except in
animals of the positive control group, and the local reactions were
recorded. Each animal was observed at least once a day for mortality and
clinical signs. Body weight was recorded once during the acclimation
period, and then on days 1 and 6.
After 2 days of resting, on day 6, the
animals received a single intravenous injection of
tritiated methyl thymidine (3H-TdR). Approximately
5 hours later, the animals were sacrificed and the auricular lymph nodes
were excised. The proliferation of lymphocytes in the lymph node
draining the application site was measured by incorporation of3H-TdR.
The results were expressed as
disintegrations per minute (dpm) per group and as dpm/node. The obtained
values were used to calculate Stimulation Indices (SI).
Mortality and clinical signs
No unscheduled deaths and no clinical
signs were observed during the observation period.
No local reactions were observed in
No notable increase in ear thickness
was observed at any tested concentrations.
The SI of the positive control was >
3; this experiment was therefore considered valid.
No notable lymphoproliferation was
noted with the test item at any tested concentrations.
The test item gave
a negative result in the murine Local Lymph Node Assay, indicative of
the absence of skin sensitization properties.
The threshold positive value of 3 for
the SI was reached in the positive control group (SI = 8.36). The
experiment was therefore considered valid.
non-irritant (increase in ear thickness < 10%).
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