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EC number: 235-985-8 | CAS number: 13080-86-9
- 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 02 - 09 May 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- adopted in 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- 4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline
- EC Number:
- 235-985-8
- EC Name:
- 4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline
- Cas Number:
- 13080-86-9
- Molecular formula:
- C27H26N2O2
- IUPAC Name:
- 4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline
Constituent 1
In chemico test system
- Details on the study design:
- TEST SYSTEM
- Supplier: JPT Peptide Technologies GmbH, Berlin, Germany
- Cysteine-containing peptide:
Amino acido sequence: Ac-RFAACAA-COOH
Batch number: 111016HS_MHeW0217
Purity: > 98%
- Lysine-containing peptide:
Amino acido sequence: Ac-RFAAKAA-COOH
Batch number: 220114HSDW_W0217
Purity: > 98%
TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
REFERENCE CONTROL (RC)
Concentration: 0.5 mM
- RC A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification.
- RC B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time.
- RC C (vehicle control) was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was the same as for the positive control because no (further) solvent was used. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally, RC C was used to calculate PPD.
CO-ELUTION CONTROL
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control.
POSITIVE CONTROL
- Substance: cinnamic aldehyde (in acetonitrile)
- Supplier: Sigma Aldrich, Steinheim, Germany
- Batch number: MKBP1014V
- Purity: > 98.4%
POSITIVE CONTROL PREPARATION
The positive control was prepared at a stock concentration of 100 mM (in acetonitrile).
TEST SUBSTANCE PREPARATION
The test substance was prepared at a stock concentration of 100 mM (in acetonitrile).
PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM
INCUBATION CONDITIONS
- Peptide solutions:
Cysteine-containing peptide: 750 µL peptide and 50 µL test item/ positive control solution and 200 µL acetonitrile
Lysine-containing peptide: 750 µL peptide and 250 µL test item/ positive control
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: 22 - 24 h
NUMBER OF REPLICATES
triplicates
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm (Agilent Technologies, Santa Clara, USA)
- Guard column: SecurityGuardTM cartridge for C18, 4.0 x 2.0 mm (Phenomenex, Torrance, USA)
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in Milli-Q water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20 (end run)
% A: 90, 75, 10, 10, 90
% B: 10, 25, 90, 90, 10
- Wavelength for detection: 220 and 258 nm
- Injection volume: 4 μL
- Column temperature: 30 °C
Results and discussion
- Positive control results:
- The positive control induced a positive response in both peptide runs, as the mean peptide depletions were 57.35% and 70.37% for lysine and cysteine, respectively.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: test substance
- Parameter:
- other: % depletion of cysteine-containing peptide (mean value of 3 replicates)
- Value:
- 1.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- (reference controls)
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: positive control
- Parameter:
- other: % depletion of cysteine-containing peptide (mean value of 3 replicates)
- Value:
- 73.6
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- (reference controls)
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Run / experiment:
- other: test substance
- Parameter:
- other: % depletion of lysine-containing peptide (mean value of 3 replicates)
- Value:
- 1.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- (reference controls)
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: positive control
- Parameter:
- other: % depletion of lysine-containing peptide (mean value of 3 replicates)
- Value:
- 58.4
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- (reference controls)
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- OTHER EFFECTS:
Precipitation was observed upon addition of the test chemical to the SPCC (Synthetic Peptide Containing Cysteine) and SPCL (Synthetic Peptide Containing Lysine) peptide solutions and in the the co-elution controls.
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, in separate experiment (do data available)
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
mean peptide concentration of reference control A and C (cysteine peptide run) = 0.503 ± 0.005 and 0.501 ± 0.010, respectively (acceptable: 0.50 ± 0.05 mM)
mean peptide concentration of reference control A and C (lysine peptide run) = 0.548 ± 0.003 and 0.546 ± 0.021, respectively (acceptable: 0.50 ± 0.05 mM)
- Acceptance criteria met for positive control (PC): yes
mean peptide depletion of the PC (cysteine peptide run) = 73.6% (range: 60.8% - 100%)
mean peptide depletion of the PC (lysine peptide run) = 58.4% (range: 40.2% - 69.0%)
- Acceptance criteria met for variability between replicate measurements: yes
SD of peptide depletion of the PC replicates (cysteine peptide run) = 1.6 (< 14.9%)
SD of peptide depletion of the test substance replicates (cysteine peptide run) = 1.1 (< 14.9%)
SD of peptide depletion of the PC replicates (lysine peptide run) = 0.4 (< 11.6%)
SD of peptide depletion of the test substance replicates (lysine peptide run) = 1.3 (< 11.6%)
Any other information on results incl. tables
Table 2: Mean peptide depletion of cysteine-containing peptide
Cysteine peptide |
|||||
Sample |
Peak area at |
Peptide |
Peptide |
Mean |
SD of |
Positive |
1002742 |
0.163 |
72.9 |
73.6 |
1.6 |
1021122 |
0.138 |
72.4 |
|||
921906 |
0.124 |
75.3 |
|||
Test |
3505855 |
0.505 |
0.0 |
1.1 |
1.1 |
3437717 |
0.495 |
1.2 |
|||
3402865 |
0.49 |
2.2 |
Table 3: Mean peptide depletion of lysine-containing peptide
Lysine peptide |
|||||
Sample |
Peak area at |
Peptide |
Peptide |
Mean |
SD of |
Positive |
1006860 |
0.225 |
54.8 |
58.4 |
0.4 |
1020611 |
0.228 |
58.2 |
|||
1024158 |
0.229 |
58.1 |
|||
Test |
2300770 |
0.532 |
2.5 |
1.1 |
1.3 |
2343450 |
0.542 |
0.7 |
|||
2367102 |
0.548 |
0.0 |
Applicant's summary and conclusion
- Interpretation of results:
- other: no skin sensitising potential based on the key event “protein reactivity”
- Conclusions:
- Under the conditions of the test, the test substance did not show reactivity towards selected peptides. However, since precipitation was observed upon addition of the test chemical to the Synthetic Peptide Containing Cysteine and Synthetic Peptide Containing Lysine peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.
In general, the result of this test alone does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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