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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))
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 5 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:
direct peptide reactivity assay (DPRA)
Details of test system:
cysteine peptide, (Ac-RFAACAA-COOH)
lysine peptide (Ac-RFAAKAACOOH)
Details on the study design:
BASIS OF THE METHOD
Skin sensitizing chemicals have the ability to covalently modify skin proteins or to be biotically or abiotically activated to become protein-reactive. Chemical-modified proteins are recognized by the immune system as foreign and trigger a specific T-cell mediated immune response. A key step in the skin sensitization process is therefore the formation of a covalent adduct between the skin sensitizer and endogenous proteins and/or peptides in the skin. Based on this wellestablished toxicity mechanism, the most straightforward approach to predict skin sensitization involves the measurement of the reactivity of a test compound towards peptides and proteins. Gerberick et al. [2, 4] therefore developed a peptide depletion assay using different heptapeptides (later coined the DPRA or ‘direct peptide reactivity assay’) to assess a chemicals ability to react with and deplete a test peptide. Depletion is measured as the loss of the peptide signal as determined by HPLC-UV.

EXPERIMENTAL DESCRIPTION
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.

HPLC-Conditions:
▪ LC-System: Agilent 1100 Series (quat. low pressure gradient pump)
▪ Column: Zorbax SB-C18, 3μm, 2.1mm x 100mm
▪ DAD-Detector: 220nm
▪ Inj.Vol.: 7μl, Temp.: 30°C, Flow: 0.35ml/min
▪ Mobile Phase: ACN + 0.085% TFA / H2O + 1% TFA
▪ Gradient: 0min: 10% ACN / 90% H2O
10min: 25% ACN / 75% H2O
11min: 90% ACN / 10% H2O
13min: 90% ACN / 10% H2O
13.5min - 20min: 10% ACN / 90% H2O (conditioning)

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

Positive control:
In each test Cinnamic aldehyde (EC: 203-213-9) (purity >99%) (supplied by Aldrich) is included as positive control.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP.

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 is described in Natsch, A., Direct peptide reactivity assay (DPRA) for skin sensitization testing: Proficiency testing at the Givaudan testing facility. Givaudan Red Corner Report, RCR 153’453, 2015.
Vehicle / solvent:
acetonitrile
Positive control:
cinnamic aldehyde
Positive control results:
The positive control gave 65.9 % depletion of the Cys-peptide and 50.7 % depletion of the Lyspeptide.
All the acceptance criteria were fulfilled for the positive control cinnamic aldehyde.
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
cysteine depletion
Value:
27.8 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
lysine depletion
Value:
1.3 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Average depletion Cys-and Lys-peptide
Value:
14.6 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Outcome of the prediction model:
low reactivity [in chemico]
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
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 5 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:
activation of keratinocytes
Details of test system:
Keratinoses transgenic cell line [442D]
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). It forms part of OECD guideline 442d.

The only feature all skin sensitizers have in common is their intrinsic electrophilicity or their potential to be metabolically transformed to electrophilic chemicals. The signaling pathway with the repressor protein Keap1(Kelch-like ECH-associated protein 1) and the transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2), which binds to the antioxidant / electrophile response element (ARE / EpRE), is known to respond to electrophilic chemicals and it was found to be a valuable cellular endpoint to detect skin sensitizers in vitro [10]. This result was confirmed by independent laboratories [11-16].

Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a final level of 1% of the solvent DMSO containing the test substance. Each compound is tested at 12 concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent control, 1 well with no cells for background value and 5 wells with a dose response of the positive control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same set-up, and a forth parallel plate is prepared for cytotoxicity determination.
After 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.
Vehicle / solvent control:
DMSO
Negative control:
not applicable
Positive control:
cinnamic aldehyde [442D]
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
Group:
test chemical
Run / experiment:
mean
Parameter:
Imax [442D]
Value:
1.87
At concentration:
1 000 other: µM
Cell viability:
The test item was strongly cytotoxic in the tested concentration range.
Mean IC50 = 13.20 µM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
EC 1.5 [442D]
Value:
7.95 µM
Cell viability:
The test item was strongly cytotoxic in the tested concentration range.
Mean IC50 = 13.20 µM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Outcome of the prediction model:
positive [in vitro/in chemico]
Other effects / acceptance of results:
Substances are rated positive if the following conditions are met:
• The Imax indicates > 1.5-fold gene induction, and this induction is statistically significant above the solvent control in a particular repetition as determined by students T-test. The EC1.5 value is below 1000 µM in all three repetitions or in at least 2 repetitions. (If the Imax is exactly equal to 1.5, the substance is still rated negative and no EC1.5 value is calculated by the evaluation sheet.)
• At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC 1.5 determining value), the cellular viability is above 70%.
• There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

Validity criteria for positive control:
Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold).
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.

Validity criteria for solvent control:
The variability of the solvent control must be below 20%.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
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.

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.
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.