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EC number: 255-410-4 | CAS number: 41496-43-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
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
- 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.
- 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 - 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 KeratinoSensTM 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 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.
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.
- 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.
- 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.
Referenceopen allclose all
The test substance was weakly cytotoxic in the tested concentration range: geometric mean IC50 = 823.96 µM (SD of 60.48 µM).
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.
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.
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