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Toxicological information

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:
November 9-26, 2015
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
no
Remarks:
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.
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: As00222569
Expiration date: May 1st, 2018

In chemico test system

Details on study design:
The test substance was freely soluble in acetonitrile at 100 mM, and this preferred solvent according the SOP could thus be used.
Positive control: Cinnamic aldehyde
Negative (vehicle) control: Acetonitrile

Test reagents: All test reagents were sourced as indicated in the standard operating procedure. The Lys-peptide (Ac-RFAAKAA, MW 775.4) was obtained from Genscript Inc. (Piscataway, NJ, USA). It has a purity of 95.6%. The Cys-peptide (Ac-RFAACAA, MW 750.1) was obtained from Genscript Inc.(Piscataway, NJ, USA). It has a purity of 96.0%.

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.

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

Results and discussion

In vitro / in chemico

Results
Key result
Parameter:
other: Average depletion Cys-and Lys-peptide
Value:
0.4
Positive controls validity:
valid
Remarks:
59.4

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The result of the DPRA assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the KeratinoSens™ assay may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of
chemicals, two congruent results in these two tests give a good prediction of the sensitizer hazard [3-5] particularly when predicting human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.

FRESKOMENTHE was non-reactive and classified into the minimal reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.
Executive summary:

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 FRESKOMENTHE 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 FRESKOMENTHE was determined by HPLC-UV.

FRESKOMENTHE was non-reactive and classified into the minimal reactivity class according to the DPRA prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.