ethyl N,S-bis(4-oxo-4-(2,6,6-trimethylcyclohex-3-en-1-yl)butan-2-yl)cysteinate

Endpoint summary

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, 2018:

GR-87-0307 (Scentaurus berry) was reactive and classified into the high reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

Keratinosens, OECD 442D, 2018:

GR-87-0307 (Scentaurus berry) was weakly toxic to the KeratinoSens™ cells. It did induce the luciferase gene above a threshold of 1.5 in all three repetitions. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2.5.2018 and 4.5.2018

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))

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:

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 reactivity assay (DPRA)

Details on the study design:

Acetonitrile (i.e. the standard solvent of the SOP) could be used for preparation of test solutions.The test was run according to the final validated SOP published by ECVAM (Dbalm protocol 154[1]).

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 well- established toxicity mechanism, the most straightforward approach to predict skin sensitization involves the measurement of the reactivity of a test compound towards peptides and proteins (reviewed in [8]). Gerberick et al. [2, 6] 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 descritpion:
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 and 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 is included as positive control.

Negative control:
Vehicle control with Acetonitrile

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 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’

Positive control results:

Cys-peptide depletion: average of measured values in the study = 66.1, standard deviation 1.1, coefficient of variation 1.6 ; criterion fulfilled
Lys-peptide depletion: average of measured values in the study = 53.6, standard deviation 2.0, coefficient of variation 3.7 ; criterion fulfilled

Key result

Parameter:

other: Average depletion Cys- and Lys-peptides

Value:

50 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

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 (not applicable and 0.6 % SD, respectively). The co-elution controls indicated no co-elution with an UV-absorbing component.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

GR-87-0307 was reactive and classified into the high reactivity class according to the prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

Executive summary:

Introduction

The Direct peptide reactivity Assay (DPRA) is anin chemicotest 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).

 

Experimental

The test substance GR-87-0307 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- 87-0307 was determined by HPLC-UV.

 

Results

GR-87-0307 was reactive and classified into the high reactivity class according to the DPRA prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2.4.2018 to 19.4.2018

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)

Version / remarks:

2014

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:

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

Details on the study design:

Positive control: Cinnamic aldehyde
Negative vehicle control: Dimethylsufoxide

All test reagents were sourced as indicated in the standard operating procedure. Luciferin was sourced from Promega.
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

Experimental Procedures:
- DMSO (i.e. the standard solvent of the SOP) could be used for preparation of test solutions. The test was run according to the final validated SOP published by ECVAM (Dbalm protocol 155).

- Basis of the method:
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. This result was confirmed by independent laboratories.

- Experimental description:
Test System(s):
The KeratinoSens™ cell line is derived from the human keratinocyte culture HaCaT. It contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene AKR1C2 [2].
The KeratinoSens™ cell line was developed by the testing lab and stored on liquid nitrogen. It was grown in 10 cm petri dishes as described in the SOP to 80% confluency prior to testing for 3 – 4 days.
Cells were counted using a counting chamber and adjusted to the desired density. During seeding into 96-well plates, the cell suspension was gently stirred and cell sedimentation was avoided by repeatedly pipetting up and down to ensure homogeneous distribution of cells.

Basic Procedure:
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.

Positive control
In each test Cinnamic aldehyde is included as positive control. It is tested in each test plate at five concentrations from 4 – 64 µM.

Endpoint & Endpoint Detection:
Two endpoints are measured: (i) Luciferase induction after a 48 h treatment with test substances and (ii) cytotoxicity as determined with the MTT assay recorded in a parallel plate with the same cell batch and made up with the same dilutions of the test substances.
Luminescence was read in a Promega Glomax Luminometer programmed to
i. add 50 µl of the luciferase substrate to each well,
ii. to then wait for 1 second and
iii. then to integrate the luciferase activity for 2 seconds.


Endpoint Value:
For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration needed to reach an 1.5-, 2- and 3- fold induction (EC1.5, EC2 and EC3) are calculated. For cytotoxicity the IC50 value is extrapolated.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the SOP. The test plates are read by a plate reader, and the generated raw data are directly pasted into this template, and all data processing is performed automatically by this Excel sheet.
For both the MTT and the luciferase data, first the background value recorded in an empty well without added cells is subtracted.
For the MTT data the % viability is then calculated for each well in the test plate in relation to the average of the six solvent control wells.
For the luciferase data the average value of the six solvent control wells is set to 1, and for each well in the test plate the fold induction is calculated in relation to this value.

The following parameters are then calculated from these processed raw data:
• Imax Maximal fold-gene induction of the luciferase gene over the full dose-response up to 1000 µM
• EC 1.5 Concentration in µM for 1.5-fold gene induction
• EC 2 Concentration in µM for 2-fold gene induction
• EC 3 Concentration in µM for 3-fold gene induction
• Pos / Neg Rating of substance according to prediction model
• reps. Positive number of independent repetitions positive / number of repetitions done
• IC50 Concentration in µM for 50% reduction of cell viability

Prediction Model
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.

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 requirement was fulfilled 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. Both criteria were fulfilled in all three repetitions. Thus all three repetitions were valid for the positive control.

Key result

Parameter:

other: EC 1.5 geometric mean

Value:

4.39 µM

Vehicle controls validity:

valid

Remarks:

negative vehicle control

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: EC 2 geometrice mean

Value:

11.17 µM

Vehicle controls validity:

valid

Remarks:

negative vehicle control

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: EC 3 geometric mean

Vehicle controls validity:

valid

Remarks:

negative vehicle control

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no induction above a given treshold

Other effects / acceptance of results:

GR-87-0307 was weakly cytotoxic in the tested concentration range.
GR-87-0307 did induce the luciferase gene above the threshold of 1.5 in all three repetitions and is thus rated as a sensitizer in the KeratinoSens™ assay.
See detailed results in the study report attached

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In all three repetitions, induction of the luciferase above the threshold of 1.5 was noted. According to the prediction model of the KeratinoSens™ assay, the test substance is rated as sensitizer.

Executive summary:

Introduction

The KeratinoSensTMassay 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).

 

Experimental

The test substance GR-87-0307 was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSensTMassay at 12 concentrations in three repetitions, each time in three replicates. After a 48 h incubation time, luciferase induction and cellular viability at each of the concentrations weredetermined.

 

 

Results

GR-87-0307 was weakly toxic to the KeratinoSens™ cells. It did induce the luciferase gene above a threshold of 1.5 in all three repetitions. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Additional information:

DPRA

A Direct peptide reactivity Assay (DPRA) was conducted to determine the reactivity of the test substance towards peptides, in accordance with the OECD Guideline 442c.

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 the tested item was determined by HPLC-UV.

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 test substance gave 100% depletion of the Cys-peptide and 0 % depletion of the Lys-peptide. The average peptide depletion is 50%. This is above the threshold of 42.47%, and the substance is thus attributed to the “high” reactivity class, rating it as a sensitizer according to the DPRA prediction model. It is therefore considered a sensitizer according to the prediction model of the DPRA.

KeratinoSens

The tested substance was submitted to the KeratinoSensTM test which a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response, following the OECD test guideline 442d.

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 tested 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 a 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

The tested sybstance was weakly toxic to the KeratinoSens™ cells. It did induce the luciferase gene above a threshold of 1.5 in all three repetitions. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay.

 

In conclusion, the tested substance suggests a classification as H317 1B based on the predictive model results.

 

Justification for classification or non-classification

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

 

No data was available for respiratory sensitisation.