[No public or meaningful name is available]

Endpoint summary

Description of key information

In vitro:

- DPRA: negative (OECD 442C, rel.1), kDPRA: negative (OECD 442C, rel. 1)

- Keratinosens: positive (OECD 442D, rel.1)

- GARD Assay: positive (eq. OECD 442E, rel.1)

 

Overall conclusion: Skin sensitiser Cat. 1B

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:

key study

Study period:

From 22 September 2021 to 23 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442C without deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

2021

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ECVAM. (2014), DB-ALM protocol 154: Direct peptide reactivity assay (DPRA) for skin sensitisation testing

Version / remarks:

21 October 2021

Deviations:

not specified

GLP compliance:

no

Remarks:

Internal study performed with the GLP spirit

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The kinetic Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the speed of the reaction of a test substance towards peptides to give a quantitative estimate of the Molecular Initiating Event in skin sensitization as an indication of sensitizer potency.
This assay has been validated for a broad range of low-molecular weight chemicals. It was found that the reaction constant derived from the assay correlates to the potency of reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated in a multi-laboratory trial and peer-reviewed by an international expert panel. It was recently implemented in OECD test guideline 442c on the Key event 1 of skin sensitization.
The kDPRA can be used for sub-classification of chemicals into GHS categories 1A and 1B/NC. In combination with evidence from the KeratinoSens and/or h-CLAT/GARD assay, it can be further used to determine a Point of Departure for risk assessment.

Details of test system:

other:

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: The Cys-peptide (Ac-RFAACAA, MW 750.9) was obtained from Genscript Inc. (Piscataway, NJ, USA). It has a purity of 98.4%.
- Preparation of the test chemical solutions: The test substance was dissolved in Acetonitrile
- Preparation of the positive controls, reference controls and co-elution controls: Not reported

INCUBATION
- Incubation conditions:
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 0.32 – 5 mM of the test chemical (dissolved in the acetonitrile fraction) in a final volume of 120 µL.

At 10, 30, 90, 150, 210 and 1440 min after the start of the incubation, the reaction is stopped by the addition of a 3 mM solution of monobromobimane (40 µL added to 120 µL incubation solution). The remaining peptide is thus fluorescently labelled after an incubation time of 5 min and can then be quantified by measuring the fluorescence using an excitation filter of 390 nm and an emission filter of 480 nm.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Cinnamic aldehyde fulfilled the acceptability criteria for the positive control. The log k (in M-1s-1) of the positive control cinnamic aldehyde at 90 min should be within the following range: -1.75 to -1.40. The measured logarithmic reaction rate at 90 min was -1.57. This value is within the acceptance range.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: log kmax

Value:

-3.5

At concentration:

5 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

Acceptance criteria for the vehicle control: The coefficient of variation of the 12 vehicle control values of a plate should be <12.5% for 5 of the 6 time points. These criteria were fulfilled (1.79 % to 4.48 % CV at the six time points)

Chemicals which are non-reactive or which have a log kmax < -2.0 are not categorised as UN GHS subcategory 1A sensitizers by the kDPRA. The measured rate constant log kmax is -3.5 (default value for non-reactive substance), therefore the test substance is not a UN GHS subcategory 1A sensitizer according to the kDPRA prediction model.

Interpretation of results:

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

Conclusions:

The test substance was non-reactive in the kDPRA. It is therefore classified into the GHS 1B/NC class, i.e. not as a strong 1A sensitizer, according to the prediction model of the kDPRA.

Executive summary:

The kinetic Direct Peptide Reactivity Assay (DPRA) study was performed according to OECD TG 442C to assess the skin sensitizing properties of the test substance.

The test substance was dissolved in acetonitrile and mixed with the Cysteine- containing peptide in five different ratios according to the standard operating procedure of the kDPRA. The Peptide depletion was monitored at six different time points by fluorescent derivatization of the parent peptide. One study with three replicates was conducted. The resulting depletion matrix vs. incubation time and test concentration was used to calculate the maximal reaction rate with the test peptide, expressed as Log k_max. The reaction rate was then used for GHS sub-classification.

The test substance was non-reactive in the kDPRA assay and thus rated with the default log k_max = -3.5 for negatives and classified as a GHS 1B/NC substance according to the kDPRA prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 May 2022 to 02 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Study performed in compliance with GLP prior to the inclusion of the GARD assay in the OECD TG 442E but is comparable to this guideline.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

30 Juin 2022

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Genomic Alergen Rapid Detection (GARD TM Skin)

Details of test system:

other: SenzaCell cell line

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical: the test item was not soluble without vehicle to facilitate the interaction. A concentration of 0.2% DMSO in well was used since the test item was not soluble in cell medium at lower DMSO concentrations. The highest soluble in well concentration of test item was 500 µM (highest permissible concentration in this assay).
- Preparation of the positive controls: 75 µM in DMSO (in-well concentration).
- Preparation of the solvent, vehicle and negative controls: 0.2% (in-well concentration)

CYTOTOXICITY ASSESSMENT:
- Conditions: the test item was exposed to a range of concentrations and was incubated for 24+/-0.5 hr at 37+/-1°C and 5+/-0.5% CO2. After incubation, the cells were harvested and stained with the viability marker Propidium Iodide and analysed by flow cytometry.
- Concentration used: from 1 to 500 µM.
- Solubility in solvents
- Solubility in incubation medium
- Results of selecting appropriate concentration and determination of cytotoxicity: the concentration chosen for main simulations was 200 µM (relative viability of 93.7%, i.e. the highest concentration within the range 84.5-95.4% as per the OECD TG 442E).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 1
- Test chemical concentrations: 200 µM in 0.2% DMSO
- Application procedure: after incubation for 24+/-0.5 hr at 37+/-1°C and 5+/-0.5% CO2, the cells were lysed in TRIzol reagent and stored at -20+/-5°C awaiting RNA isolation.
RNA was isolated from the lysed cells according to the manufacturer's instruction. An RNA sample aliquot was taken from each RNA quantification and quality control. Both aliquots and RNA sample were stored at -80°C.
- Exposure time
- Study evaluation and decision criteria used: Decision value (DV) ≥ 0 <=> Skin sensitiser / DV < 0 <=> non-sensitiser.
- Description on study acceptance criteria:
1/ Solubility: the test item should be soluble by ocular inspection, in a GARDskin compatible vehicle to a minimum in-well concentration of 1 µM
2/ Phenotypic quality controls: the cells should pass the phenotypic quality control on the day of cell stimulation.
3/ The cell viability of the Main stimulations should have fulfilled the following acceptance criteria
The positive control and a test item expected to induce cytotoxicity should have a relative viability of 84.5-95.4%.
The negative control and a test item not expected to induce cytotoxicity should have a relative viability or ≥ 95.5%.
The unstimulated control should have an absolute viability ≥ 84.5%
4/ RNA quality control: a total RNC concentration ≥ 20 ng/nL and a RIN value ≥8.
5/ Endpoint measurements quality controls: the RCC files generated from the nCounter MAX analysis files should fulfil the following criteria:
Image Quality > 0.75
Linearity > 0.95
Limit of Detection (LOD): POS_E / LOD > 1
Binding density (BD): 0.05
QUANTIFICATION:
- Analysis system: nCounter MAX Digital Analyser

Vehicle / solvent control:

DMSO

Negative control:

other: unstimulated control

Positive control:

other: p-Phenylenediamine (PPD)

Positive control results:

Sensitiser, Acceptance criteria: Pass (cf. attached study report).

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Decision value (DV)

Value:

4.93

Vehicle controls validity:

valid

Negative controls validity:

valid

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item is classified as a sensitizer in the GARDskin assay.

Executive summary:

The Genomic Alergen Rapid Detection assay (GARD TM Skin) study was performed similarly to OECD TG 442E to assess the skin sensitizing hazard properties of the test item.

The test item was dissolved in DMSO and then added to the cells. For the cytotoxicity assessment, stock solutions in a concentration range were prepared by serial dilutions and the final applied concentration ranged from 500 µM to 1 µM. Cells were incubated with the test item under standard conditions. After exposure, cells were stained with propidium iodide and cell viability was measured by PFAS analysis. Cytotoxicity was observed and the in-well concentration used endpoint classification was therefore 200 µM.

Following cellular stimulations, RNA was isolated and endpoint measurements were performed using the GARDskin GPC. All samples passed the standard acceptance criteria for the GARDskin assay.

With a mean Decision Value of 4.93 (>0), the test item is classified as a sensitizer in the GARDskin assay when tested at an in-well concentration of 200 µM.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 August 2021 to 06 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442C without deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

2021

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ECVAM. (2014), DB-ALM protocol 154: Direct peptide reactivity assay (DPRA) for skin sensitisation testing

Version / remarks:

21 October 2021

Deviations:

not specified

GLP compliance:

no

Remarks:

Internal study performed with the GLP spirit

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of a test 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 approachfor testing and assessment (IATA). It was recently also implemented in OECD test guideline 497 on Defined Approaches for skin sensitisation testing.

Details of test system:

other:

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: The Lys-peptide (Ac- RFAAKAA, MW 775.9) was obtained from Genscript Inc. (Piscataway, NJ, USA). It has a purity of 95.6%. The Cys-peptide (Ac-RFAACAA, MW 750.9) was obtained from Genscript Inc. (Piscataway, NJ, USA). It has a purity of 98.4%.
- Preparation of the test chemical solutions: The test substance was dissolved in Acetonitrile
- Preparation of the positive controls, reference controls and co-elution controls: Not reported

INCUBATION
- Incubation conditions:
(1) Lys-peptide: 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.
(2) Cys-peptide: 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.
- Precipitation noted: No precipitation noted

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Yes
- Verification of the suitability of the HPLC for test chemical and control substances: Yes

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Cinnamic aldehyde fulfilled the acceptability criteria for the positive control. The results are presented in Table 7.4.1/3 and 7.4.1/4 in the study report attached.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.5 %

At concentration:

25 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

1.8 %

At concentration:

5 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Not applicable
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for reference controls A to C: Yes. Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (1.3 % and 0.7 % SD, respectively).
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes. The co-elution controls indicated no co-elution with an UV-absorbing component.
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: as per the OECD TG 442C

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test substance 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 DPRA.

Executive summary:

The Direct Peptide Reactivity Assay (DPRA) study was performed according to OECD TG 442C to assess the first key event in the skin sensitization adverse outcome pathway.

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 test substancewas determined by HPLC-UV.

The test substance gave 1,8 %depletion of the Cys-peptide and 0.5 % depletion of the Lys-peptide. The average peptide depletion is 1.1 %. This is below the threshold of 6.38%, and the substance is thus attributed to the “minimal” reactivity class, rating it as a non-sensitizer according to the DPRA prediction model.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 September 2021 to 07 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442D without deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM DB-ALM protcol 155: KeratinoSens(TM) protocol

Version / remarks:

2014

Deviations:

no

GLP compliance:

no

Remarks:

Internal study performed with the GLP spirit

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

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.
The assay underwent validation at the European Centre for the Validation of Alternative Methods (ECVAM) and an OECD test guideline was adopted (OECD TG 442d) . The protocol was published as DB-ALM protocol 155. The assay was proposed by ECVAM to be used as part of an integrated approach for testing and assessment (IATA) . It was recently also implemented in OECD test guideline 497 on defined approaches for skin sensitisation testing.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: As per DB-ALM Protocol n°155
- Preparation of the test chemical serial dilutions: twelve concentrations in the range from 0.98 to 2000 µM
- Preparation of the positive controls: 64μM, 32μM, 16μM, 8μM, 4μM in DMSO.
- Preparation of the solvent, vehicle and negative controls: DMSO 1%
- Stable dispersion obtained: yes
- Other: NA

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 µM
- Solubility in solvents: The test substance was freely soluble in DMSO at 200 mM
- Solubility in incubation medium: Not reported
- Cytotoxicity assessment performed: Yes
- Final concentration range selected on basis of: cytotoxicity

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: Three
- Number of repetitions: One
- Test chemical concentrations: 0.98 to 1000 µM
- Application procedure: As per the DB-ALM Protocol n°155
- Exposure time: 48 hours
- Study evaluation and decision criteria used: As per the DB-ALM Protocol n°155

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): not stated
- Incubation conditions: As per DB-ALM Protocol n°155
- Washing conditions: As per DB-ALM Protocol n°155
- Precipitation noted: None

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: Promega Glomax Luminometer
- Plate used: Not reported
- Lysate preparation: Assumed to be as per DB-ALM Protocol n°155

DATA EVALUATION
- Cytotoxicity assessment: PrestoBlue® assay
- Prediction model used: 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 su bstance 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.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

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. In all three experiments performed here both criteria were fulfilled. Thus all three repetitions were valid for the positive control.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

1.97 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:

IC50 [442D]

Value:

2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

3.81

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:

OTHER EFFECTS:
- Visible damage on test system: none seen

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes (cf. "positive controls results")
- Range of historical values if different from the ones specified in the test guideline: as per the guideline
- Overall outcome: 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. This conclusion is also clearly supported by the analysis of the dose-response curve with overall clear induction of the luciferase reporter gene to be observed.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

GR-87-6331 was weakly toxic to the KeratinoSens™ cells. In all three repetitions, it did induce the
luciferase gene above the threshold of 1.5. It is therefore considered a sensitizer according to the
prediction model of the KeratinoSens™ assay. Analysis whether the results falls within the borderline
range indicates that the result is not borderline.

Executive summary:

The study was performed according to the Keratinosens assay, as described in OECD TG 442D and DB- ALM Protocol No. 155 to assess the second key even in the skin sensitizing adverse outcome pathway.

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

 

The test substance was weakly toxic to the KeratinoSens™ cells. In all three repetitions, it did induce the luciferase gene above the threshold of 1.5. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay. Analysis whether the results falls within the borderline range indicates that the result is not borderline.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In chemico/in vitro studies

DPRA: negative (OECD 442C, rel.1):

The Direct Peptide Reactivity Assay (DPRA) study was performed according to OECD TG 442C to assess the first key event in the skin sensitization adverse outcome pathway.

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 test substancewas determined by HPLC-UV.

The test substance gave 1,8 %depletion of the Cys-peptide and 0.5 % depletion of the Lys-peptide. The average peptide depletion is 1.1 %. This is below the threshold of 6.38%, and the substance is thus attributed to the “minimal” reactivity class, rating it as a non-sensitizer according to the DPRA prediction model.

kDPRA: negative (OECD 442C, rel. 1)

The kinetic Direct Peptide Reactivity Assay (DPRA) study was performed according to OECD TG 442C to assess the skin sensitizing properties of the test substance.

The test substance was dissolved in acetonitrile and mixed with the Cysteine- containing peptide in five different ratios according to the standard operating procedure of the kDPRA. The Peptide depletion was monitored at six different time points by fluorescent derivatization of the parent peptide. One study with three replicates was conducted. The resulting depletion matrix vs. incubation time and test concentration was used to calculate the maximal reaction rate with the test peptide, expressed as Log k_max. The reaction rate was then used for GHS sub-classification.

The test substance was non-reactive in the kDPRA assay and thus rated with the default log k_max = -3.5 for negatives and classified as a GHS 1B/NC substance according to the kDPRA prediction model.

Keratinosens: positive (OECD 442D, rel.1)

The study was performed according to the Keratinosens assay, as described in OECD TG 442D and DB- ALM Protocol No. 155 to assess the second key even in the skin sensitizing adverse outcome pathway.

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

The test item was weakly toxic to the KeratinoSens™ cells. In all three repetitions, it did induce the luciferase gene above the threshold of 1.5. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay. Analysis whether the results falls within the borderline range indicates that the result is not borderline.

 

GARD Assay: positive (eq. OECD 442E, rel.1)

The Genomic Alergen Rapid Detection assay (GARD TM Skin) study was performed similarly to OECD TG 442E to assess the skin sensitizing hazard properties of the test item.

The test item was dissolved in DMSO and then added to the cells. For the cytotoxicity assessment, stock solutions in a concentration range were prepared by serial dilutions and the final applied concentration ranged from 500 µM to 1 µM. Cells were incubated with the test item under standard conditions. After exposure, cells were stained with propidium iodide and cell viability was measured by PFAS analysis. Cytotoxicity was observed and the in-well concentration used endpoint classification was therefore 200 µM.

Following cellular stimulations, RNA was isolated and endpoint measurements were performed using the GARDskin GPC. All samples passed the standard acceptance criteria for the GARDskin assay.

With a mean Decision Value of 4.93 (>0), GR-87-6331 is classified as a sensitizer in the GARDskin assay when tested at an in-well concentration of 200 µM.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the 2 out of 3 Defined Approach (2o3 DA), the test substance is determined to be a skin sensitizer due to the negative DPRA study and positive Keratinosens and GARD studies.

Following an independent peer review, the kDPRA was considered to be scientifically valid to discriminate UN GHS subcategory 1A skin sensitisers from those not categorised as 1A (non-subcategory 1A)
according to UN GHS. The kDPRA can therefore be used as a follow-up test
method for sub-categorisation of chemicals identified as UN GHS Category 1 skin
sensitisers, or on its own by using positive results for direct classification of a
chemical into UN GHS subcategory 1A, depending on the regulatory framework. Looking at the kDPRA study result here, log Kmax is below -2.0 therefore the test substance is not categorised as GHS UN subcategory 1A.

In conclusion, the test substance is classified as skin sensitizer category 1B (H317) according to GHS UN and CLP regulations.