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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to
Guideline:
other: ECVAM (2014), DB-ALM protocol 154: Direct peptide reactivity assay (DPRA) for skin sensitization testing
Principles of method if other than guideline:
​Although the in vitro test was not carried out in a registered GLP laboratory, the Givaudan in vitro Technologies laboratory based at it's Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP. In addition this Givaudan laboratory at Dubendorf devised the KeratinoSens testing protocol and instigated the assay acceptance with ECHA and the OECD. The Givaudan laboratory at Dubendorf also was a member of the DPRA acceptance testing program of laboratories and has over 4 years experience with both the KeratinoSens and DPRA assays.
GLP compliance:
no
Remarks:
Conducted within the "spirit" of GLP (See Principles of method if other than guideline)
Type of study:
other: Direct peptide reactivity assay (DPRA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Trivial name : Hexenyl-3-Cis Isobutyrate
Expiration date : 03.03.2016
Batch number : Ve00318809
Physical form : Liquid

In chemico test system

Details on study design:
The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).

The test substance HEXENYL-3-CIS-ISOBUTYRATE was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by HEXENYL-3-CIS-ISOBUTYRATE was determined by HPLC-UV.

Test System(s):
The Lys-peptide Ac-RFAAKAA is incubated at a final concentration of 0.5 mM in an ammonium acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold excess of the test substance (25 mM) dissolved in acetonitrile.
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of a 10-fold excess of the test substance (5 mM) dissolved in acetonitrile.

Endpoint & Endpoint Detection:
24 h after start of the incubation the remaining peptide is quantified with HPLC-UV.

Endpoint Value:
The endpoint is expressed as % peptide depletion.

Positive control
In each test Cinnamic aldehyde is included as positive control.

Prediction Model
Based on the average peptide depletion values for both the Cysteine and the Lysine peptide, a reactivity class is attributed to the substances. Average depletion below 6.38% indicates minimal reactivity, substances in this class are predicted as non-sensitizers by the DPRA.
The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is described in Test report RCR 153’453, ‘Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility’.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Cys-peptide depletion
Value:
32.7
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Lyspeptide depletion
Value:
0.8
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Average peptide depletion
Value:
16.7
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (1.7% and 0.5% SD, respectively).
The co-elution controls indicated no co-elution with an UV-absorbing component.

Any other information on results incl. tables

When using HPLC-UV analysis only, HEXENYL-3-CIS-ISOBUTYRATE led to 32.7% depletion of the Cys-peptide. It was therefore considered reactive and classified into the LOW reactivity class according to the classical DPRA prediction model.

When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation rather than adduct formation. Thus, based on this more sophisticated and detailed analysis the test chemical is not directly peptide reactive and the assay does not indicate sensitization potential for Hexenyl-3-cis-isobutyrate.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
When using HPLC-UV analysis only, HEXENYL-3-CIS-ISOBUTYRATE led to 32.7% depletion of the Cys-peptide. It was therefore considered reactive and classified into the LOW reactivity class according to the classical DPRA prediction model.
When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation rather than adduct formation. Thus, based on this more sophisticated and detailed analysis the test chemical is not directly peptide reactive and the assay does not indicate sensitization potential for Hexenyl-3-cis-isobutyrate.
Executive summary:

When using HPLC-UV analysis only, HEXENYL-3-CIS-ISOBUTYRATE led to 32.7% depletion of the Cys-peptide. It was therefore considered reactive and classified into the LOW reactivity class according to the classical DPRA prediction model.

When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation rather than adduct formation. Thus, based on this more sophisticated and detailed analysis the test chemical is not directly peptide reactive and the assay does not indicate sensitization potential for Hexenyl-3-cis-isobutyrate.