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Additional toxicological data

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Administrative data

Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Publication intended to develop an alternative in vitro method for sensitization, test substance used as example of non-sensitizer.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Type of study / information:
Covalent modification of skin proteins by electrophiles is a key event in the induction of skin sensitisation but not skin irritation although the exact nature of the binding mechanisms has not been determined empirically for the vast majority of sensitisers. It is also unknown whether immunologically relevant protein targets exist in the skin contributing to effecting skin sensitisation.
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of this work was to verify for selected non-sensitisers that no protein haptenation occurs even under forcing conditions. The test substance solubilised in 3:2 dimethylsulphoxide: water (v/v). Samples were prepared using solutions of test chemicals and solution of human serum albumin (HAS) at a 1:100 and 1:1000 molar ratio of protein: chemical in 50 mM ammonium acetate. Samples were incubated at 37 ℃. Incubation length varied between 24 h and 28 days, depending on the experiment.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzaldehyde
EC Number:
202-860-4
EC Name:
Benzaldehyde
Cas Number:
100-52-7
Molecular formula:
C7H6O
IUPAC Name:
benzaldehyde
Details on test material:
Batch No.: not specified
Purity: > 95% pure

Results and discussion

Any other information on results incl. tables

HSA peptide mapping and assessment of experimental variability:

On average, more than 90% of the native HSA sequence was observed in any one MALDI-ToF spectrum of the control samples. Relative signal intensities changed depending on the position of the laser beam on the sample-matrix mixture as well as laser intensity at the time of data acquisition, thus only semiquantitative information can be derived when MALDI-MS spectra are compared. Although observing reduced signal intensity could potentially mean that the relevant peptide is modified by a hapten, covalent adducts could reliably be detected only by appearance of new signals with mass shifts of modified peptides corresponding to hapten adducts.

Analyses of samples incubated with the test substance:

MALDI-MS analyses of the HSA-the test substance samples did not show the appearance of any new signals when compared to the control sample spectrum. Additionally, all samples were subjected to RP-HPLC separation and rigorous ES-MS/MS analyses of the collected fractions failed to produce any evidence of covalent modifications of HSA by the test substance.

Applicant's summary and conclusion

Conclusions:
It was concluded that the test substance do not covalently modify HSA in this test system under any incubation conditions. This is indicative for the absence of haptenation of the test substance, which is expected for a non sensitizer.
Executive summary:

The aim of this work was to verify for selected non-sensitisers (the test substance is considered to be a non-sensitizer by the authors of the report) that no protein haptenation occurs even under forcing conditions (binding to any of the nucleophilic centra in intact human serum albumin (HSA)). The test substance solubilised in 3:2 dimethylsulphoxide: water (v/v). Samples were prepared using solutions of test chemicals and solution of human serum albumin (HSA) at a 1:100 and 1:1000 molar ratio of protein: chemical in 50 mM ammonium acetate (as HSA has 125 nucleophilic side chains the molar ratios of nucleophile: electrophile are effectively 1:0.8 and 1:8, respectively). Samples were incubated at 37 ℃. Incubation length varied between 24 h and 28 days, depending on the experiment.

It was concluded that the test substance do not covalently modify HSA in this test system under any incubation conditions. This is indicative for the absence of haptenation of the test substance, which is expected for a non sensitizer (although in theory reaction with nucleophils via Schiff base formation may be possible for the test substance).