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EC number: 208-706-2 | CAS number: 538-93-2
- 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 vitro
- Remarks:
- Direct Peptide Reactivity Assay (DPRA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Jan - Feb 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Certificate produced by "Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht" Rheinland Pfalz
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:
- 2015
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- Isobutylbenzene
- EC Number:
- 208-706-2
- EC Name:
- Isobutylbenzene
- Cas Number:
- 538-93-2
- Molecular formula:
- C10H14
- IUPAC Name:
- (2-methylpropyl)benzene
- Test material form:
- liquid
- Details on test material:
- - batch no.: EXP-18/11/346
- State of aggregation: liquid
- Log KOW: 3.94 (calculated); 4.8 at 23°C and pH 6.0 (measured)
- Molecular weight: 134.22 g/mol
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch No.: EXP-18/11/346
- Purity test date: 18/11/2015
- Log Kow: 3.94 (calculated); 4.8 at 23°C and pH 6.0 (measured)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM (considering a molecular weight of 134.22 g/mol and a purity/contents of 99.9%) preparation in acetonitrile within 4 hours of preparation of test-substance samples. After short stirring the test substance was soluble in the vehicle. All test-substance preparations and dilutions were made in glass vials.
FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in acetonitrile
In vitro test system
- Details on the study design:
- TEST SYSTEM
- Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol); Lysine-(K-)containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K containing peptide). The peptide stock solution were used for preparing the calibration samples, the test-substance and control samples.
- Source: The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and/or RS Synthesis, Louisville KY, USA and/or JPT Peptide Technologies GmbH, Berlin, Germany) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
CONTROLS
- Vehicle control: acetonitrile: Set A) performance control (analyzed together with the calibration samples without incubation); Set B) Stability control (placed at the very start and ending of the sample list for HPLC analysis); Set C) for calculation of the peptide depletion (analyzed with the samples)
- Positive control: ethylene glycol dimethacrylate (EGDMA; CAS no. 97-90-5) (prepared as a 50 mM solution in acetonitrile)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide
VEHICLE
- Vehicle: acetonitrile
- Reason for choice of the vehicle: The test substance was soluble in acetonitrile
SAMPLE PREPARATION
- Peptide stock solutions were mixed with the test substance or positive control or vehicle control at a ration of 1:10 (0.5 mM C-peptide, 5 mM test substance) or 1:50 (0.5 mM K-peptide, 25 mM test substance)
EXPERIMENTAL PROCEDURE
- No. of replicates: 3 (for each peptide)
- The test substance was prepared at a 100 mM concentration. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containg peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).
- Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis
- Samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours
- The remaining non-depleted peptide concentration was determined by HPLC with gradient elution and UV-detection at 220 nm about 24 hours after sample preparation (for details on HPLC conditions see tab. 3). The analysis time itself did not exceed 30 hours.
- Calibration samples of known concentration (dissolved in 20% acetonitrile in the respective buffer), prepared from the respective peptide stock solution used for test-substance incubation were measured before analysis of the test-substance samples with the same analytical method (for details see tab. 1)
DATA EVALUATION (for detailed formulas see "Any other information on material and methods")
Calculation of the peptide concentrations:
- For each peptide a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration. The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).
Calculation of the peptide depletion:
- The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance. When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.
ACCEPTANCE CRITERIA
- The standard calibration curve should have an r² >0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- The positive control should cause depletion of both peptides comparable to historic data.
Results and discussion
In vitro / in chemico
Results
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: inconclusive
- Remarks:
- due to the limited solubility of the test substance
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see tab. 9a+b
Any other information on results incl. tables
Table 6: Peptide depletion for C-peptide
Reaction with cysteine- peptide |
peptide depletion [%] |
|||
sample 1 |
sample 2 |
sample 3 |
mean; SD |
|
NC: ACN |
2.35 |
-3.88 |
1.52 |
0.00; 3.38 |
Test substance |
-3.92 |
-3.19 |
0.29 |
-2.27; 2.25 |
PC: EGDMA in ACN |
63.85 |
67.91 |
71.14 |
67.63; 3.65 |
Table 7: Peptide depletion for K-peptide
Reaction with lysine-peptide |
peptide depletion [%] |
|
|||
sample 1 |
sample 2 |
sample 3 |
mean; SD |
||
NC: ACN |
0.23 |
-0.49 |
0.27 |
0.00; 0.43 |
|
Test substance |
-0.93 |
-0.66 |
0.40 |
-0.40; 0.70 |
|
PC: EGDMA in ACN |
13.80 |
13.63 |
13.50 |
13.64; 0.15 |
Table 8: Mean peptide depletions
|
Cysteine-Peptide
mean depletion [%] SD [%] |
Lysine-Peptide
mean depletion [%] SD [%] |
mean of both depletions [%] |
||
Test substance |
-2.27 |
2.25 |
-0.40 |
0.70 |
0.00 |
PC: EGDMA in ACN |
67.63 |
3.65 |
13.64 |
0.15 |
40.64 |
Table 9a: Historic control data of vehicle control (acetonitrile) (not including present study)
C-peptide concentration [mM] | K-peptide concentration [mM] | |
Min | 0.451 | 0.486 |
Max | 0.511 | 0.536 |
Mean | 0.484 | 0.508 |
SD | 0.015 | 0.011 |
n | 42 | 40 |
Table 9b: Historic control data of positive control (EGDMA, 50 mM in acetonitrile) (not including present study)
C-peptide concentration [mM] | C-peptide depletion [%] | K-peptide concentration [mM] | K-peptide depletion [%] | |
Min | 0.144 | 43.32 | 0.395 | 6.79 |
Max | 0.282 | 70.07 | 0.491 | 21.52 |
Mean | 0.216 | 55.20 | 0.445 | 12.90 |
SD | 0.032 | 6.23 | 0.020 | 3.10 |
n | 32 | 30 |
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Remarks:
- limited solubility of the test substance leading to emulsive samples
- Conclusions:
- Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.
- Executive summary:
The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.
The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides.
Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed (K-containing peptide, only) and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm (both peptides).
The following results were obtained in the DPRA:
The test substance was soluble in acetone at a concentration of 100 mM. The samples of the test substance and the peptide stock solutions were emulsions at the time of preparation and after 24 hours.
No co-elution of test substance and peptides was present. However, for the C-peptide reaction an alternative analysis method was used.
The mean C-peptide depletion, caused by the test substance was determined to be -2.27%.
The mean K-peptide depletion, caused by the test substance was determined to be -0.40%.
Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.
Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.
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