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Administrative data

Description of key information

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.

DPRA

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 peptides.

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 chosen.

LuSens

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.

Key value for chemical safety assessment

Skin sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

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 markers.

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 peptide depletion.

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.

 
 Test Method  Test Result  Test Evaluation
 Direct Peptide Reactivity Assay (DPRA)  0.31% mean peptide depletion (-2.24% cysteine-peptide depletion; 0.63% lysine-peptide depletion).  Negative
 Keratinocyte Activation Assay - LuSens  In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed  Negative
 Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)  Not determined  Not determined

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification 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.