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EC number: 701-116-0
CAS number: 2156592-45-7
LLNA (OECD 429, Harlan 2012): sensitizing
a GLP compliant study according to OECD guideline 429 the possible skin
sensitisation potential of (Z)-4-[C11-13
(branched) alkylamino]-4-oxo-2-butenoic acid
was studied (Harlan CCR, 2012). Three groups each of five female mice
were treated once daily with the test item at concentrations of 2.5, 5,
and 10% (w/w) in methyl ethyl ketone by topical application to the
dorsum of each ear for three consecutive days. The test item could be
dissolved in the vehicle. The appropriateness of the used concentrations
was previously assessed by two pre-experiments. A control group of five
mice was treated with the vehicle (methyl ethyl ketone) only. Five days
after the first topical application the mice were injected intravenously
into a tail vein with radio-labelled thymidine (³H-methyl thymidine).
Approximately five hours after intravenous injection, the mice were
sacrificed, the draining auricular lymph nodes were excised, pooled per
animal and immediately weighed. Furthermore, after excision of the lymph
nodes, both ears of the mice were punched at the apical area using a
biopsy punch and were immediately weighed pooled per animal using an
analytical balance. Afterwards, single cell suspensions of lymph node
cells were prepared from lymph nodes pooled per animal. An aliquot of
each cell suspension was used for determination of lymph node cell
count. Subsequently the suspensions were washed and incubated with
trichloroacetic acid overnight. The proliferative capacity of pooled
lymph node cells was determined by the incorporation of ³H-methyl
thymidine measured in a β-scintillation counter. The animals did not
show any signs of systemic toxicity during the course of the study and
no cases of mortality were observed. On application day 2 and 3, the
animals treated with test item concentrations of 5 and 10% showed an
erythema of the ear skin (Score 1). On day 4, only the animals of the
high dose group showed an erythema of the ear skin (Score 1). A
statistically significant increase in ear weights was observed in the
high dose group in comparison to the vehicle control group (p<0.05).
However, this was considered to be not biologically relevant, as the
observed increase did not exceed the threshold value of 25% for
excessive local skin irritation mentioned in OECD guideline 429.
Furthermore, for BALB/c mice, a cutoff-value of 1.1 was reported for a
positive response of the ear weight index regarding ear skin irritation.
None of the indices determined for the test item treated groups exceeded
this threshold. A test item is regarded as a sensitiser in the LLNA if
exposure to one or more test item concentration results in a 3-fold or
greater increase in incorporation of ³HTdR compared with concurrent
controls, as indicated by the Stimulation Index (S.I.). The estimated
test item concentration required to produce a S.I. of 3 is referred to
as the EC3 value. In this study Stimulation Indices (S.I.) of 3.42,
8.41, and 14.20 were determined with the test item at concentrations of
2.5, 5, and 10% (w/w) in methyl ethyl ketone, respectively. An EC3 value
could not be calculated because all obtained Stimulation Indices
exceeded the threshold value of 3 for a positive response. An outlier
was identified in the vehicle control group (DPM values determined for
animal number 1). However, as exclusion of the outlier only slightly
changed the mean value of the vehicle group and had no influence on the
overall test result, the value in question was not excluded from
calculation. A statistically significant and biologically relevant
increase in DPM value, lymph node weight and –cell count was obtained in
all test item treated groups in comparison to the vehicle control group
(p<0.05) and a clear dose response was observed. Furthermore, the
cutoff-value for a positive response regarding the lymph node cell count
index of 1.55 reported for BALB/c mice was exceeded in all dose groups
(indices of 2.08, 3.55, and 4.94). The test item (Z)-4-[C11-13
(branched) alkylamino]-4-oxo-2-butenoic acid
was a skin sensitiserunder
the test conditions of this study.
According to the findings of the available studies, the test substance
has to be classified R43 (Directive 67/548/EEC) and Skin sensitisation
Cat. 1 (CLP). However based on the available data, no subcategorization
into 1A/1B is possible.
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