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Description of key information

The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the DPRA may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of substances, two congruent results in these two tests give a good prediction of the sensitizer hazard, in particular when comparing against human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.

In all three repetitions, no induction of the luciferase above the threshold of 1.5-fold was noted. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as nonsensitizer. This is also clearly supported by the analysis of the dose-response curve in Figure 4 with overall no induction of the luciferase reporter gene to be observed.

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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: As00222569
Expiration date: May 1st, 2018
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.
Key result
Parameter:
other: Average depletion Cys-and Lys-peptide
Value:
0.4
Positive controls validity:
valid
Remarks:
59.4
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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 16-26, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
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:
activation of keratinocytes
Specific details on test material used for the study:
Name (as stated in the report): Freskomenthe
Batch: As00222569
Expiration date: May 1st, 2018
Details on study design:
The test substance was freely soluble in DMSO at 200 mM, and this preferred solvent according the
SOP could thus be used.
Positive control: Cinnamic aldehyde
Negative (vehicle) control: Dimethylsulfoxide
Test reagents:The Luciferase substrate was prepared according to the following recipe: 20 mM Tricine; 2.67 mM MgSO4; 0.1 mM EDTA; 33.3 mM DTT; 270 μM Coenzyme A; 470 μM Luciferin; 530 μM ATP; pH 7.8
Positive control results:
EC 1.5 average (Induction values Reference): 15.36
Key result
Parameter:
other: Cytotoxicity determinations
Remarks:
Geometric Mean IC 50 (μM)
Value:
> 1 000
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Luciferase induction
Remarks:
Average IMAX (fold induction)
Value:
1.06
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing and assessment (IATA)[9]. A parallel test in the DPRA may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of substances, two
congruent results in these two tests give a good prediction of the sensitizer hazard [5, 7, 18], in particular when comparing against human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.

In all three repetitions, no induction of the luciferase above the threshold of 1.5-fold was noted. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as nonsensitizer. This is also clearly supported by the analysis of the dose-response curve in Figure 4 with overall no induction of the luciferase reporter gene to be observed.
Executive summary:

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response. This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to 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 DMSO and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

FRESKOMENTHE was non-toxic to the KeratinoSens™ cells up to 1000 μM. In all three repetitions, it did not induce the luciferase gene above a threshold of 1.5-fold. It is therefore considered a nonsensitizer according to the prediction model of the KeratinoSens™ assay.

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

Justification for classification or non-classification

Frenskomenthe did not elicit any sensitization reaction under the test conditions, therefore it does not need to be classified according to GHS criteria.