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EC number: - | CAS number: -
- 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:
- 2017-12-01 to 2018-01-20
- 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- February 04, 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
Test material
- Reference substance name:
- 4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]
- Cas Number:
- 1115233-52-7
- Molecular formula:
- C21 H18 Cl F
- IUPAC Name:
- 4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]
- Test material form:
- solid
Constituent 1
In chemico test system
- Details of test system:
- cysteine peptide, (Ac-RFAACAA-COOH)
- lysine peptide (Ac-RFAAKAACOOH)
- Details on the study design:
- PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: 20.32 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.48 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. 20.56 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.09 mL) to reach a concentration of 0.667 mM. All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.
- Preparation of the test chemical solutions: The test item was freshly prepared immediately prior to use. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. In a pre-experiment the test item was not soluble in acetonitrile, dist. water, dist. water : acetonitrile 1:1 (v/v), isopropanol, methanol, 1,4-butanediol. The test item was completely soluble in N,N-dimethylformamide(DMF), therefore, DMF was chosen as suitable vehicle for the main experiments.
- Preparation of the positive controls, reference controls and co-elution controls:
Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100mM was prepared and was included in every assay run for both peptides.
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 220nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference Control: Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C 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 prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
INCUBATION
- Incubation conditions: The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in table 1 (see “Any other information on materials and methods”).
- Precipitation noted:
For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis.
PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20 % acetonitrile : 80 % buffer ( v / v ) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions in the range from 0.0 to 0.534 mM.
- Verification of the suitability of the HPLC for test chemical and control substances: Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50 % phase A and 50 % phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10 % to 25 % acetonitrile over 10 minutes, followed by a rapid increase to 90 % acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. For details on HPLC system refer to “Any other information on materials and methods”.
DATA EVALUATION
- Cys and Lys peptide detection wavelength: λ = 220 nm
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 mean depletion of both peptides was 62.40 %.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Group:
- test chemical
- Run / experiment:
- mean
- Parameter:
- mean cystein depletion
- Value:
- 5.58
- At concentration:
- 100 mM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Group:
- test chemical
- Run / experiment:
- mean
- Parameter:
- mean lysine depletion
- Value:
- 0.21
- At concentration:
- 100 mM
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Outcome of the prediction model:
- other: Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not applicable
DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified
ACCEPTANCE OF RESULTS:
Please refer to table 8 and 9 under “Any other information on results”.
Any other information on results incl. tables
Table 2: Cysteine and Lysine Values of the Calibration Curve
Sample | Cysteine Peptide | Lysine Peptide | ||
Peak Area | Peptide Concentration [mM] | Peak Area | Peptide Concentration [mM] | |
STD1 | 5221.6792 | 0.5340 | 4254.6284 | 0.5340 |
STD2 | 2648.5273 | 0.2670 | 2161.7087 | 0.2670 |
STD3 | 1327.5009 | 0.1335 | 1051.8925 | 0.1335 |
STD4 | 670.7400 | 0.0667 | 525.2503 | 0.0667 |
STD5 | 334.8562 | 0.0334 | 260.5259 | 0.0334 |
STD6 | 166.3505 | 0.0167 | 130.8325 | 0.0167 |
STD7 | 0.0000 | 0.0000 | 0.0000 | 0.0000 |
Table 3: Depletion of the Cysteine Peptide
Cysteine Peptide | ||||||
Sample | Peak Area | Peptide Conc. [mM] | Peptide Depletion [%] | Mean Peptide Depletion [%] | SD of Peptide Depletion [%] | CV of Peptide Depletion [%] |
Positive Control | 1609.4324 | 0.1633 | 68.11 | 68.17 | 0.05 | 0.08 |
1604.4860 | 0.1628 | 68.21 | ||||
1605.4248 | 0.1629 | 68.19 | ||||
Test Item | 4722.0444 | 0.4814 | 4.97 | 5.58 | 0.54 | 9.77 |
4682.2471 | 0.4774 | 5.77 | ||||
4670.3384 | 0.4761 | 6.01 |
Table 4: Depletion of the Lysine Peptide
Lysine Peptide | ||||||
Sample | Peak Area | Peptide Conc. [mM] | Peptide Depletion [%] | Mean Peptide Depletion [%] | SD of Peptide Depletion [%] | CV of Peptide Depletion [%] |
Positive Control | 1675.9452 | 0.2100 | 57.93 | 56.62 | 1.13 | 2.00 |
1753.5808 | 0.2197 | 55.98 | ||||
1754.5223 | 0.2198 | 55.95 | ||||
Test Item | 4024.7078 | 0.5039 | 0.13 | 0.21 | 0.15 | 72.44 |
4025.2893 | 0.5039 | 0.12 | ||||
4014.3962 | 0.5026 | 0.39 |
Table 5: Prediction Model 1
Cysteine 1:10/ Lysine 1:50 Prediction Model 1
Mean Cysteine andLysine PPD | Reactivity Class | DPRA Prediction² |
0.00% ≤ PPD ≤ 6.38% | No or Minimal Reactivity | Negative |
6.38% < PPD ≤ 22.62% | Low Reactivity | Positive |
22.62% < PPD ≤ 42.47% | Moderate Reactivity | |
42.47% < PPD ≤ 100% | High Reactivity |
1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.
2 DPRA predictions should be considered in the framework of an IATA.
Table 6: Prediction Model 2
Cysteine 1:10 Prediction Model
Cysteine PPD | ReactivityClass | DPRA Prediction² |
0.00% ≤ PPD ≤ 13.89% | No or Minimal Reactivity | Negative |
13.89% < PPD ≤ 23.09% | Low Reactivity | Positive |
23.09% < PPD ≤ 98.24% | Moderate Reactivity | |
98.24% < PPD ≤ 100% | High Reactivity |
Table 7: Categorization of the Test Item
Prediction Model | Prediction Model 1 | Prediction Model 2 | ||||
Test Substance | Mean Peptide Depletion [%] | Reactivity Category | Prediction | Mean Peptide Depletion [%] | Reactivity Category | Prediction |
Test Item | - | - | - | 5.58 | Minimal Reactivity | negative |
Positive Control | 62.40 | High Reactivity | positive | 68.17 | Moderate Reactivity | positive |
Table 8: Acceptance Criteria for Cysteine Peptide
Cysteine Peptide Run | |||
Acceptance Criterion | Range | Value | pass/fail |
coefficient of determination | R² >0.99 | 0.9999 | pass |
mean peptide concentration of RC A | 0.45 ≤ x ≤ 0.55 mM | 0.5193 | pass |
mean peptide concentration of RC C (PC) | 0.45 ≤ x ≤ 0.55 mM | 0.5147 | pass |
mean peptide concentration of RC C (TI) | 0.45 ≤ x ≤ 0.55 mM | 0.5066 | pass |
CV of the peak area of RC B | <15 % | 0.44 | pass |
CV of the peak area of RC C (PC) | <15 % | 0.53 | pass |
CV of the peak area of RC C (TI) | <15 % | 0.34 | pass |
mean peptide depletion of the PC | 60.8 % < x < 100 % | 68.17 | pass |
SD of peptide depletion of the PC replicates | <14.9 % | 0.05 | pass |
SD of peptide depletion of the TI replicates | <14.9 % | 0.54 | pass |
Table 9: Acceptance Criteria for Lysine Peptide
Lysine Peptide Run | |||
Acceptance Criterion | Range | Value | pass/fail |
coefficient of determination | R² >0.99 | 0.9999 | pass |
mean peptide concentration of RC A | 0.45 ≤ x ≤ 0.55 mM | 0.5003 | pass |
mean peptide concentration of RC C (PC) | 0.45 ≤ x ≤ 0.55 mM | 0.4987 | pass |
mean peptide concentration of RC C (TI) | 0.45 ≤ x ≤ 0.55 mM | 0.5045 | pass |
CV of the peak area of RC B | <15 % | 0.48 | pass |
CV of the peak area of RC C (PC) | <15 % | 0.47 | pass |
CV of the peak area of RC C (TI) | <15 % | 0.32 | pass |
mean peptide depletion of the PC | 40.2 % < x < 69.0 % | 56.62 | pass |
SD of peptide depletion of the PC replicates | <11.6 % | 1.13 | pass |
SD of peptide depletion of the TI replicates | <11.6 % | 0.15 | pass |
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Conclusions:
- In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.
- Executive summary:
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.
In the present study, according to OECD TG 442C, the test item was dissolved in N.N-dimethylformamide (DMF) based on the results of the pre-experiments. Based on a molecular weight of 324.82 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.
For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. After the 24 h± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item precipitation was observed when diluted with the lysine peptide solution. After the 24 h± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis.
A minor co-elution of the test item with the lysine peptide peak was observed. The peak area determined in the co-elution controls corresponds to 0.0104 mM of lysine peptide. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.
The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of the cysteine peptide was ≤ 13.89 % (5.58 %). According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.
Since precipitation was observed for the lysine and cysteine peptide samples containing also test item after the incubation period, no final prediction can be made.
In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.
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