Registration Dossier

Diss Factsheets

Administrative data

Description of key information

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 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.

DPRA assay, OECD 442C, 2017:

Scentaurus Clean (GR-86 -6599) was reactive with the Cys-peptide (27.8% depletion) and classified into the LOW reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

Keratinosens, OECD 442D, 2017:

Scentaurus Clean (GR-86 -6599 was strongly toxic to the KeratinoSens cells

In all three repetitions, it did induce the luciferase gene above a threshold of 1.5 at 8 μM and in two occasions this induction occurred at a

non-toxic dose. It is therefore considered a skin sensitizer according to the prediction model of the KeratinoSens™ assay.

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:
7 to 10 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
no
Remarks:
GLP equivalent
Type of study:
direct peptide reactivity assay (DPRA)
Details on the 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 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 was determined by HPLC-UV.
Key result
Parameter:
other: Average depletion Cys-and Lys-peptide
Value:
14.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: % (unit of average depletion)
Other effects / acceptance of results:
The test substance gave 27.8 % depletion of the Cys-peptide and 1.3 % depletion of the Lyspeptide.
The average peptide depletion is 14.6 %. This is above the threshold of 6.38%, and the substance is thus attributed to the “LOW” reactivity class, rating it as a sensitizer according to the DPRA prediction model. Direct reactivity with the Cys-peptide was also verified by an LC-MS based analysis of the reaction sample, indicating formation of a novel peptide adduct with the mass of 1046.7 indicating direct reaction of the test chemical (MW 296) with the Test Peptide (MW 750.5).

Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (3.7 % and 1.9 % SD, respectively). The co-elution controls indicated no co-elution with an UV-absorbing component.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
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 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.
GR-86-6599 was reactive with the Cys-peptide (27.8% depletion) and classified into the LOW reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA. Additional analysis with LC-MS indicated that it indeed formed a covalent adduct with the test peptide.
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 GR-86-6599 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 GR-86-6599 was determined by HPLC-UV.

GR-86-6599 was reactive with the Cys-peptide (27.8% depletion) and classified into the LOW reactivity class according to the prediction model. It is therefore considered a 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:
16 to 20 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
no
Remarks:
GLP equivalence
Type of study:
activation of keratinocytes
Details on the study design:
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 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.
Positive control results:
Cinnamic aldehyde was run in all three repetitions. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in all three repetitions. The induction at 64 μM and the EC 1.5 for cinnamic aldehyde were also calculated. The targets are: (i) Average induction in the three replicates for cinnamic aldehyde at 64 μM should be between 2 and 8, and (ii) the EC 1.5 value should be between 7 μM and 30 μM. At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed here both criteria were fulfilled in all three repetitions. Thus all three repetitions were valid for the positive control.
Key result
Run / experiment:
other: 1
Parameter:
other: Average IMAX (fold induction)
Value:
1.87
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
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, induction of the luciferase above the threshold of 1.5 was noted, in two of them at non-cytotoxic concentrations. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as a sensitizer. This conclusion is also supported by the analysis of the dose-response curve with overall dose-dependent induction of the luciferase reporter gene just below the cytotoxic concentration 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 GR-86-6599-43 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.

GR-86-6599-43 is strongly toxic to the KeratinoSens™ cells. In all three repetitions, it did induce the luciferase gene above a threshold of 1.5 at 8 μM and in two occasions this induction occurred at a non-toxic dose. It is therefore considered a skin sensitizer according to the prediction model of the KeratinoSens™ assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance is classified as skin sensitiser in Category 1 (H317: May cause an allergic skin reaction) according to the CLP and to the GHS.

 

No data was available for respiratory sensitisation.