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EC number: 203-678-8
CAS number: 109-53-5
A combination of the following three in vitro methods, addressing key
events of the adverse outcome pathway (AOP) for skin sensitization
(OECD, 2012) as defined by the OECD, were part of the in vitro Skin
Sensitization Turnkey Testing Strategy:
• protein reactivity (DPRA),
• activation of keratinocytes (LuSens), and
• activation of dendritic cells (h-CLAT).
However, in the current case for Isobutylvinylether the results derived
with DPRA and LuSens were sufficient for a final assessment. Therefore
further testing in h-CLAT was waived.
The reactivity of Isobutylvinylether towards synthetic cysteine (C)- or
lysine (K)-containing peptides was evaluated in the Direct Peptide
Reactivity Assay (DPRA). For this purpose, the test substance was
incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the
remaining non-depleted peptide concentrations were determined by high
performance liquid chromatography (HPLC) with gradient elution and
UV-detection at 220 nm.
The test substance was dissolved at 100 mM in acetonitrile. One valid
test run was performed. Three samples of the test substance were
incubated with each peptide in ratios of 1:10 (for C-containing peptide)
or 1:50 (for K-containing peptide). Additionally, triplicates of the
concurrent vehicle control (= VC) were incubated with the peptides.
Further, in order to detect possible interference of the test substance
with the peptides, a co-elution control was performed and the samples
were analyzed by measuring UV absorbance at 258 nm in order to calculate
the area ratio 220 nm / 258 nm.
The following results were obtained in the DPRA: the test substance was
dissolved in acetonitrile at a concentration of 100 mM. The samples of
the test substance with the peptides were solutions at the time of
preparation. Visual observation after the 24-hour incubation time did
not reveal precipitates in any samples of the test substance with the
No co-elution of test substance and peptides was present.
The mean C-peptide depletion, caused by the test substance was
determined to be -2.24%.
The mean K-peptide depletion, caused by the test substance was
determined to be 0.63%.
Negative depletions were considered to be “zero” for calculation of the
mean peptide depletion, which was thus calculated to be 0.31%.
Based on the observed results and applying the cysteine 1:10 / lysine
1:50 prediction model it was concluded that Isobutylvinylether shows
minimal or no chemical reactivity in the DPRA under the test conditions
The keratinocyte activating potential of Isobutylvinylether was
evaluated in the LuSens assay. For this purpose, the test substance was
incubated with a luciferase reporter cell line (LuSens cells) for ca. 48
hours at 37°C and antioxidant response element (ARE) dependent
luciferase activity was measured in a luminometer.
In order to determine the concentrations suitable for the main
experiment a pre-test (non-GLP) was performed. Cells were exposed to
several concentrations of the test substance and cytotoxicity was
determined by MTT assay. No cytotoxicity was observed.
In the main test luciferase activity was measured after 48-hour
exposure. In parallel a MTT assay was performed to assess cytotoxicity
of the test substance. A total of 2 valid experiments were performed.
The following results were observed:
At concentrations used in the main experiment the test substance was
soluble in DMSO (100 x stock preparations) and in 1% DMSO in culture
medium 3 (final concentrations). No precipitates were noticed in any
concentration after 48 hours.
In summary, after 48 hours of exposure to test substance
Isobutylvinylether luciferase activity in LuSens cells was not induced
in at least two consecutive concentrations with statistical significance
affording at least 70% viability in at least two independent
experiments. From this it has to be concluded that Isobutylvinylether
does not have a keratinocyte activating potential.
Based on the results, Isobutylvinylether is not considered peptide
reactive and does not activate keratinocytes. Applying the evaluation
criteria, Isobutylvinylether is predicted not to be a skin sensitizer.
The in vitro skin sensitization turnkey strategy was initiated with the
DPRA and the LuSens.
As an unambiguous result was obtained after these two tests, no further
test was performed.
Direct Peptide Reactivity Assay (DPRA):
Chemical reactivity has been shown to be well associated with allergenic
potency (Gerberick et al., 2007) and has been described as the molecular
initiating event in the OECD adverse outcome pathway (OECD Publication
No.168; ENV/JM/MONO(2012)10). Within this context measuring the amount
of proteins with nucleophilic side chains such as cysteine or lysine
residues after incubation with putative allergens serves as surrogate
In the DPRA the reactivity of a test item towards synthetic cysteine
(C)- or lysine (K)-containing peptides is evaluated. For this purpose,
the test substance is incubated with synthetic peptides for 24 hours and
the remaining non-depleted peptide concentration is determined
thereafter by high performance liquid chromatography with gradient
elution and UV-detection at 220 nm.
The peptide depletion of test-substance incubated samples is compared to
the peptide depletion of the NC samples and expressed as relative
ARE Reporter Assay (LuSens):
Keratinocyte activation has been identified as one of the key events of
the adverse outcome pathway for skin sensitization as identified by the
OECD (OECD Publication No.168; ENV/JM/MONO (2012)10). The LuSens assay
is an in vitro method for the identification of keratinocyte activating
substances using the genetically modified keratinocytes (LuSens, Bauch
et al. 2012 and Ramirez et al. 2014, and 2016). It employs the reporter
gene for luciferase under the control of an antioxidant response element
(ARE) and hence monitors Nrf-2 transcription factor activity. The
endpoint measurement is the up-regulation of the luciferase activity
after 48 hours incubation with test substances. This up-regulation is an
indicator for the activation of the Keap1/Nrf2/ARE signaling pathway.
for sensitisation is not warranted according to the criteria of EU
Classification, Labelling and Packaging of Substances and Mixtures (CLP)
Regulation (EC) No. 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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