[No public or meaningful name is available]

Endpoint summary

Description of key information

In vitro:

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

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

Overall conclusion: Not skin sensitizer

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:

From 17 February to 11 March 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 775.4) 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 95.6%. A reference sample of the Cys-peptide dimer (MW 1499.2) was obtained from Genscript Inc, it has a purity of 90.2%.
- 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.
The positive control gave 71.2 % depletion of the Cys-peptide when measured with the LC-MS method.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.3 %

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:

24.2 %

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 (0.9 % 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

When using HPLC-UV analysis only, the test substance led to 24.2% depletion of the Cys-peptide and 1.3% of the Lys-peptide and an average peptide depletion of 12.7%. It was therefore classified into the LOW reactivity class according to the classical DPRA prediction model.
When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation / dimer formation rather than adduct formation / direct reactivity. No indication for a peptide adduct was found in this analysis. For the positive control, direct adduct formation could be verified indicating direct reactivity for the positive control. Thus, based on this more sophisticated and detailed analysis, the test chemical is not directly peptide reactive, but can only trigger peptide dimerization which is not considered itself as a molecular initiating event in skin sensitization.
Since this LC-MS based prediction model is not part of the validated DPRA protocol, this additional analysis should be used in a weight-of-evidence based analysis of the sensitization potential of a test chemical.

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 24.2 % depletion of the Cys-peptide and 1.3 % depletion of the Lyspeptide. The substance is thus attributed to the “LOW” reactivity class according to the DPRA prediction model., rating it as a non-sensitizer according to the DPRA prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 28 September 2020 to 02 October 2020

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 was run in both repetitions. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This requirement was fulfilled in both 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 both experiments performed here both criteria were fulfilled. Thus both repetitions were valid for the positive control.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

0 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

107.7 µM

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:

Imax [442D]

Value:

1.23

Vehicle controls validity:

valid

Negative controls validity:

not applicable

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 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 both repetitions, no 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 non-sensitizer.

Interpretation of results:

GHS criteria not met

Conclusions:

In both repetitions, no 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 non-sensitizer.

Executive summary:

The study was performed according to the Keratinosens assay, as described in OECD TG 442D and DB- ALM Protocol No. 155.

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 moderatly toxic to the KeratinoSens™ cells. In both independent 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)

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 24.2 % depletion of the Cys-peptide and 1.3 % depletion of the Lyspeptide. The substance is thus attributed to the “LOW” reactivity class according to the DPRA prediction model., rating it as a non-sensitizer according to the DPRA prediction model.

 

Keratinosens: negative (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.

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 moderatly toxic to the KeratinoSens™ cells. In both independent 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.

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) as defined in the OECD guideline 497, the test substance is determined to not skin sensitizing due to the negative DPRA study and negative Keratinosens study.

It is therefore not classified as skin sensitizer according to GHS UN and CLP regulations.