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

Endpoint summary

Description of key information

Three tests are available for the test substance:

- DPRA (according to OECD 442c): negative

- Keratinosens (according to OECD 442d): weakly positive

- QSAR (OASIS TIMES-LMC, Skin sensitization with autoxidation model): test substance predicted as weak skin sensitizer and its metabolites are not predicted to be skin sensitizers.

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 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.

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.

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

Positive control results:

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

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

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

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

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. 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 19, 2021 to April 23, 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. 442D but Not GLP.

Qualifier:

according to guideline

Guideline:

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

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:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

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

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

- 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.
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.

- Negative/Solvent control
In each test Dimethylsulfoxide is included as negative/solvent control. It is tested in each test plate at 1%.

- 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.

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 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:

EC 1.5 [442D]

Value:

75.75 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:

Imax [442D]

At concentration:

1 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Imax = 9.03

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

The test substance was weakly cytotoxic in the tested concentration range: geometric mean IC50 = 823.96 µM (SD of 60.48 µM).

Interpretation of results:

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

Conclusions:

The result of the KeratinoSens™ assay should be used as part of a Defined Approach (DA) or 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. This approach was implemented as the “2 out of 3” DA in OECD test guideline 497.

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 strong induction of the luciferase reporter gene to be observed.

Executive summary:

Introduction

The KeratinoSens^TM 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).

 

Experimental

The test substance 2-methyl-3-(p-tolyl)propionaldehyde was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSens^TM 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.

 

Results

2-methyl-3-(p-tolyl)propionaldehyde was weakly toxic to the KeratinoSens™ cells. In all three repetitions, it did induce the luciferase gene above a threshold of 1.5. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay. 

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OASIS TIMES v.2.30.1.11

2. MODEL (incl. version number)
Skin sensitization with autoxidation v.24.29

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS number: 41496-43-9
SMILES: CC(Cc1ccc(C)cc1)C=O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please see the attached QMRF report

5. APPLICABILITY DOMAIN
Please see the attached QPRF report

6. ADEQUACY OF THE RESULT
Please see the attached QPRF report

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6, May/July 2008

GLP compliance:

no

Specific details on test material used for the study:

SMILES: CC(Cc1ccc(C)cc1)C=O

Key result

Group:

test chemical

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

the test substance is predicted to be a weak skin sensitizer by QSAR

Outcome of the prediction model:

other: the test substance is predicted to be a weak skin sensitizer by QSAR

Other effects / acceptance of results:

This QSAR prediction is considered to be valid, because the substance was in the applicability domain of the model.

The test substance was predicted to be a weak skin sensitizer with the Skin sensitization with autoxidation model of OASIS TIMES. The metabolites of the test substance were not predicted to be sensitizers.

Interpretation of results:

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

Conclusions:

The test substance was predicted to be a weak skin sensitizer with the Skin sensitization with autoxidation model of OASIS TIMES. The metabolites of the test substance were not predicted to be sensitizers.
As a Weight of Evidence approach, taking into consideration the negative DPRA assay and the weakly positive Keratinosens test, the test substance is considered a weak skin sensitizer and a classification of Skin sensit. Cat 1B is warranted as a worst-case scenario.

Executive summary:

A QSAR was conducted using the model "Skin sensitization with autoxidation" of the software OASIS TIMES. The QSAR program calculated a weak sensitization potential of the test substance. This QSAR prediction is considered to be valid, because the substance was in the applicability domain of the model.

As a Weight of Evidence approach, taking into consideration the negative DPRA assay and the weakly positive Keratinosens test, the test substance is considered a weak skin sensitizer and a classification of Skin sensit. Cat 1B is warranted as a worst-case scenario.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Due to contradictory results following DPRA and Keratinosens assays, a QSAR prediction was conducted using OASIS TIMES-LMC software (especially the Skin sensitization with autoxidation model) in order to clarify the skin sensitization potential of the test substance and to conclude on the classification.

The DPRA test did not indicate that the test substance was skin sensitizer while the Keratinosens assay showed that the test substance was weakly toxic to the keratinocytes. The QSAR prediction indicated that the test substance was predicted to be a weak skin sensitizer (but not its metabolites). The prediction was considered valid since the test substance was in the applicability domain of the model.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the test substance is considered to be a weak skin sensitizer. As a consequence, in a worst-case scenario, a classification as Skin sensit. 1B is warranted under the EU CLP Regulation No 1272/2008.