2-methyl-3-(p-tolyl)propionaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From July 25, 2021 to September 06, 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed according to OECD test guideline No. 442C , but not GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

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 its Dubendorf site in Switzerland performs all in vitro studies within the "spirit" of GLP.

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-3-(p-tolyl)propionaldehyde

EC Number:

255-410-4

EC Name:

2-methyl-3-(p-tolyl)propionaldehyde

Cas Number:

41496-43-9

Molecular formula:

C11H14O

IUPAC Name:

2-methyl-3-(4-methylphenyl)propanal

Test material form:

liquid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

- 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.

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

- Positive control
In each test Cinnamic aldehyde is included as positive control.

- Negative control
In each test Acetonitrile is included as negative 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.
In case co-elution occurs with the Lysine peptide only, an alternative prediction model is proposed by the test guideline. In this Cysteine only model, chemicals are rated as sensitizers if they lead to >13.89% depletion of the Cysteine peptide.

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.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

All the acceptance criteria were fulfilled for the positive control cinnamic aldehyde.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

11.1 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

-1.2 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Mean peptide depletion rate

Value:

5 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference controls: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Acceptance criteria met for the calibration curve: Yes
- Range of historical values if different from the ones specified in the test guideline: see Appendix C of the report attached

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

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.

2-methyl-3-(p-tolyl)propionaldehyde 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 the DPRA. However, 2-methyl-3-(p-tolyl)propionaldehyde is an aldehyde, and reactive aldehydes do not always react with the Lys-peptide under DPRA conditions, thus this result should be considered in a weight-of-evidence approach.

Executive summary:

Introduction

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).

 

Experimental

The test substance 2-methyl-3-(p-tolyl)propionaldehyde 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 2-methyl-3-(p-tolyl)propionaldehyde was determined by HPLC-UV.

 

Results

2-methyl-3-(p-tolyl)propionaldehyde was non-reactive and classified into the minimal reactivity class according to the DPRA prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.