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EC number: 202-818-5 | CAS number: 100-09-4
- 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:
- 28 Nov - 02 Dec 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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:
- The Department of Health of the Governement of the United Kindgom, Medicines & Healthcare products Regulatory Agency (MHRA)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- p-anisic acid
- EC Number:
- 202-818-5
- EC Name:
- p-anisic acid
- Cas Number:
- 100-09-4
- Molecular formula:
- C8H8O3
- IUPAC Name:
- 4-methoxybenzoic acid
Constituent 1
In chemico test system
- Details on the study design:
- The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
TEST SYSTEM
- Supplier of synthetic peptides: Anaspec
- Peptide stock solution preparation: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
Peptide calibration standrad preparation: Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.
VEHICLE CONTROL
- Substance: acetonitrile
POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as 100 mM solution in acetonitrile. A 100 mM stock solution of the test item in acetonitrile/water 90/10 v/v was prepared.
CO-ELUTION CONTROL
- Co-elution controls were set up in parallel to sample preparation without respective peptide solution to verify whether a test chemical co-elutes with the cysteine or lysine peptide.
REFERENCE CONTROLS
Stability controls (Reference Control B and Reference Control C) and precision controls of both peptides were prepared at a concentration of 0.5 mM. Reference Control B samples and the precision control sample contained acetonitrile. The reference control C was prepared with propan-2-ol replacing acetonitrile.
TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.
INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: minimum of 22 h
NUMBER OF REPLICATES
for each peptide triplicates were prepared for test item and control C; control B samples were prepared in 6-fold
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Analytical Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Pre-column: Phenomenex, AJO-4286
- HPLC mobile phase:
A: 0.1% trifluoracetic acid in water
B: 0.085% trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Column temperature: 30 °C
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20; MP A (%): 90, 75, 10, 10, 90, 90; MP B (%): 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Injection volume: 2 μL (slow draw rate)
- Retention time cysteine: approximately 11 minutes
- Retention time lysine: approximately 7 minutes
Results and discussion
- Positive control results:
- The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The peptide depletion of the positive control for the cysteine peptide was 71.7%. The mean peptide depletion of the positive control for the lysine peptide was 58.8%.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: mean of three runs
- Parameter:
- other: mean cysteine depletion (%)
- Value:
- 1.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- since the cysteine depletion is ≤ 6.38 the prediction for sensitisation is considered negative
- Key result
- Run / experiment:
- other: mean of three runs
- Parameter:
- other: mean lysine depletion (%)
- Value:
- 1.01
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- since the lysine depletion is ≤ 6.38 the prediction for sensitisation is considered negative
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The laboratory has demonstrated technical proficiency for the DPRA by successfully testing the 10 profiency chemicals outlined in the OECD 442C guideline
ACCEPTANCE CRITERIA:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent positve control value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine depletion,
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine depletion,
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is in the range of 0.45 to 0.55 mM.
Both peptide runs and the test item results met the acceptance criteria of the test.
Any other information on results incl. tables
Table 2. Overall achieved depletion values
Test item |
Cysteine peptide depletion (%) |
Lysine peptide depletion (%) |
Overall mean depletion (%) |
Reactivity class |
DPRA prediction |
p-anisic acic |
1.40 |
1.01 |
1.20 |
No to minimal |
Negative |
Table 3. Cysteine peptide depletion
Sample |
Peak area (µV.sec) |
Peptide concentration1 |
Peptide Depletion (%) |
Mean Depletion (%) |
SD |
Positive control |
236103 |
105.70 |
71.52 |
71.7 |
0.08 |
234831 |
105.13 |
71.62 |
|||
235271 |
105.33 |
71.62 |
|||
p-anisic acic |
821285 |
369.43 |
1.433 |
1.40 |
0.43 |
825232 |
371.21 |
0.9533 |
|||
818128 |
368.01 |
1.813 |
SD Standard Deviation
1 Samples prepared at a concentration of 376 µg/mL (0.5 mM)
2 Calculated against a mean Reference Control B area of 827610 µV.sec (n=6)
3 Calculated against a mean Reference Control C area of 833170 µV.sec (n=3)
Table 4. Lysine peptide depletion
Sample |
Peak area (µV.sec) |
Peptide concentration1(µg/mL) |
Peptide Depletion (%) |
Mean Depletion (%) |
SD |
Positive control |
317845 |
158.71 |
59.12 |
58.8 |
0.29 |
322285 |
160.93 |
58.52 |
|||
319492 |
159.53 |
58.82 |
|||
p-anisic acic |
777172 |
388.56 |
-0.2093 |
1.01 |
1.78 |
751867 |
375.89 |
3.053 |
|||
774140 |
387.03 |
0.1823 |
SD Standard Deviation
1 Samples prepared at a concentration of 376 µg/mL (0.5 mM)
2 Calculated against a mean Reference Control B area of 776330 µV.sec (n=6)
3 Calculated against a mean Reference Control C area of 775550 µV.sec (n=3)
Applicant's summary and conclusion
- Interpretation of results:
- other: no skin sensitising potential based on the key event "direct peptide binding" of skin sensitisation AOP
- Conclusions:
- Under the conditions of the test, it can be concluded, that the test substance is not predicted a sensitiser in the Direct Peptide Binding Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.
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