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EC number: 600-039-9 | CAS number: 10023-48-0
- 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
QSAR prediction: negative
Skin sensitisation (OECD 442C): positive
Skin sensitisation (OECD 442D): positive
WoE conclusion from in vitro skin sensitisation battery: skin sensitising
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:
- 07 - 22 Apr 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- adopted 05 Feb 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:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- other: (in chemico) reactivity against synthetic peptides with a thiol or amino group
- 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 METHOD
The direct peptide reactivity assay (DPRA) is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, the protein reactivity. The reativity of the substance towards model synthetic peptides containing either lysine or cysteine is quantified. In the DPRA the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance is quantified. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 and 258 nm. Cysteine- and lysine peptide depletion values are then calculated and used in the prediction model, which assigns the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
TEST SYSTEM
- Supplier of synthetic peptides: JPT Peptide Technologies GmbH
- Peptide stock solution preparation: Stock solutions of each peptide were prepared by dissolution of pre-weighed aliquots of the approprate peptide in appropriate buffer solution.
Cysteine-containing peptide: 18.48 mg cysteine was dissolved in 36.584 mL phosphate buffer (pH 7.5).
- Concentration: 0.667 mM
Lysine-containing peptide: 18.82 mg lysine was dissolved in 35.159 mL ammonium acetate buffer (pH 10.2).
- Concentration: 0.667 mM
VEHICLE CONTROL
- Substance: water
- Justification for selecting vehicle: the test substance was soluble in the vehicle.
POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as 100 mM solution in acetonitrile.
CO-ELUTION CONTROL
- Co-elution controls were set up in parallel to sample preparation without respective peptide solution to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide.
REFERENCE CONTROLS
- Acetonitrile was used to verify the accuracy of the calibration curve for peptide quantification (Reference contol A) and to verify the stability of the respective peptide over the analysis time (Reference control B)
- Water and acetonitrile were used to verify that the solvents do not impact the percent peptide depletion (Reference controls C)
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 ± 2.5 °C
- Duration of treatment / exposure: minimum of 24 ± 2 h
NUMBER OF REPLICATES
for each peptide triplicates were prepared for treatment substance and controls
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent 1200 Series, with Chemstation, Rev. B.04.01
- Analytical Column: Agilent Zorbax SB-C18, 3.5 µm, 100 x 2.1 mm
Pre-column: Phenomenex, AJO-4286, 4.0 x 2.0 mm
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Column temperature: 30 °C
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution
- Injection volume: 10 μL
- peptide standards: calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, and a buffer blank were measured in parallel with the test substance samples - Positive control results:
- The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean peptide depletion of the positive control for the cysteine peptide was between 60.8% and 100% (74.90 %). The mean peptide depletion of the positive control for the lysine peptide was between 40.2% and 69.0% (54.93%).
- Key result
- Run / experiment:
- other: cysteine run
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 52.81
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- 74.90
- Key result
- Run / experiment:
- other: lysine run
- Parameter:
- other: mean peptide depletion [%]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Remarks:
- In the lysine run co-elution of the test substance and the lysine peptide due to a shift of the lysine peptide peak was observed.
- Other effects / acceptance of results:
- OTHER EFFECTS:
In the lysine run a a co-elution of the test substance with the lysine peptide peak was observed. Since the reference control samples allowed clear identification of the lysine peptide and since the test item did not elute between 6.5 and 7.7 min, it was clear that the lysine peptide peak had shifted and that the peak area of that peak did only represent remaining lysine peptide. As all validity criteria were fulfilled the results of the lysine run was not considered having an influence on the quality or validity of the overall results.
ACCEPTANCE CRITERIA
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) 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
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.
Both peptide runs and the test item results met the acceptance criteria of the test. - Interpretation of results:
- other: skin sensitising potential based on the key event "protein reactivity"
- Conclusions:
- Under the conditions of the test, the test substance showed reactivity towards selected proteins. 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 is required.
- Endpoint:
- skin sensitisation, other
- Remarks:
- (Q)SAR prediction
- Type of information:
- (Q)SAR
- Adequacy of study:
- weight of evidence
- 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
Lhasa Limited Derek Nexus
2. MODEL (incl. version number)
Derek Nexus: 5.0.1 and Nexus: 2.1.0
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Smiles Code: CC1=C(CCOP([O-])(O)=O)SC=[N+]1CC2=CN=C(C)N=C2N
CAS: 10023-48-0
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: Structural alerts
5. APPLICABILITY DOMAIN
- Descriptor domain: The target chemical falls within the applicability domain of the prediction.
6. ADEQUACY OF THE RESULT
Within Derek, there are over 504 alerts covering a wide range of toxicological endpoints. An alert consists of a toxicophore (a substructure known or thought to be responsible for the toxicity) and is associated with literature references, comments and examples. The toxicity predictions are the result of two processes. The program first checks whether any alerts in the knowledge base match toxicophores in the query structure. The reasoning engine then assesses the likelihood of a structure being toxic. There are nine levels of confidence: certain, probable, plausible, equivocal, doubted, improbably, impossible, open, contradicted. The reasoning model considers the following information: a) the toxicological endpoint; b) the alerts that match toxicophores in the query structure; c) the physico-chemical property values calculated for the query structure; and d) the presence of an exact match between the query structure and a supporting example within the knowledge base. - Principles of method if other than guideline:
- Calculation based on Derek Nexus: 5.0.1 and Nexus: 2.1.0. QSAR prediction of the skin sensitisation potential of the test substance.
- GLP compliance:
- no
- Remarks on result:
- no indication of skin sensitisation
- Interpretation of results:
- other: the prediction results was negative for skin sensitising potential, based on QSAR prediction (DEREK Nexus). The results may only be used for classification purposes together with other data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 09 - 24 May 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Version / remarks:
- adopted in Feb 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- activation of keratinocytes
- Details on the study design:
- TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance by addressing the second molecular key event of the Adverse Outcome Pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement compared with the respective solvent controls is used to support the discrimination between skin sensitiser and non-sensitisers.
TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 6 (Experiment 1), 8 (Experiment 2)
CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-pyruvate, 10% fetal bovine calf serum and 1% geneticin (final concentration 500 µg/mL)
Assay medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-g lucose and Na-pyruvate and 10% fetal bovine calf serum
Test substance exposure medium: Dulbecco’s Modified Eagle Medium (GlutaMAX™) supplemented with 1.0 g/L D-glucose and Na-Pyruvate and 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0
TEST CONCENTRATIONS
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000 and 2000 μM
CONTROLS
Solvent control:
- Substance: DMSO
Positive control:
- Substance: cinnamic aldehyde
- Final concentration: 4 - 64 μM
EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0
NUMBER OF REPLICATIONS: triplicates in two independent experiments
DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 600 nm
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0
DETERMINATION OF LUMINESCENCE
- Cell lysis reagent: Luciferase Cell Culture Lysis 5x Reagent Kit (Promega, Cat. No. E1531, Lot No. 000087405)
- Luciferase reagent: Luciferase Assay Kit (Promega, Cat. no. E1501, Lot. No. 0000237533)
- Device: plate reader - Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.28 in experiment 1; 2.65 in experiment 2).
- Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: luciferase activity
- Value:
- 3.76
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: cell viability [%]
- Value:
- 79.6
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: EC1.5 [µM]
- Value:
- 810.73
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: luciferase activity
- Value:
- 2.75
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: cell viability [%]
- Value:
- 66
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: EC1.5 [µM]
- Value:
- 268.21
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The average coefficient of variation of the luminescence reading for the vehicle control was < 20% (19.7% in and 12.5% in Experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.28 and 2.65 in Experiment 1 and 2, respectively) and the calculated EC1.5 value was between 7 and 34 µM (32.87 and 15.08 µM in Experiment 1 and 2, respectively).
In the first experiment, a max luciferase activity (Imax) induction of 3.76 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 79.6%. The further concentrations showed no significant luciferase induction >1.5. The calculated EC1.5 was < 1000 µM (810.73 µM).
In the second experiment, a max luciferase activity (Imax) induction of 2.75 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 66.0%. The lowest tested concentration with a significant luciferase induction >1.5 (1.70) was found to be 500 µM. The corresponding cell viability was >70% (71.9%). The calculated EC1.5 was < 1000 µM (268.21 µM). - Interpretation of results:
- other: skin sensitising potential based on the key event “inflammatory response in keratinocytes”
- Conclusions:
- Under the conditions of the test, the test substance has a keratinocyte activating potential. 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 is required.
Referenceopen allclose all
Table 1: Cysteine and Lysine Values of the Calibration Curve
Sample |
Cysteine Peptide |
Lysine Peptide |
||
Peak Area |
Peptide Concentration [mM] |
Peak Area |
Peptide Concentration [mM] |
|
STD7 |
0.0000 |
0.0000 |
0.0000 |
0.0000 |
STD6 |
153.8068 |
0.0167 |
139.6547 |
0.0167 |
STD5 |
321.6256 |
0.0334 |
281.4845 |
0.0334 |
STD4 |
642.2243 |
0.0667 |
555.3300 |
0.0667 |
STD3 |
1318.5444 |
0.1335 |
1100.1805 |
0.1335 |
STD2 |
2593.4377 |
0.2670 |
2176.1533 |
0.2670 |
STD1 |
5075.1016 |
0.5340 |
4280.0898 |
0.5340 |
Table 2: Depletion of the Cysteine Peptide
Cysteine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Concentration [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1193.1283 |
0.1239 |
74.99 |
74.90 |
0.25 |
0.34 |
1187.7434 |
0.1233 |
75.10 |
||||
1210.7732 |
0.1258 |
74.62 |
||||
Test Item |
2310.8784 |
0.2412 |
51.07 |
52.81 |
1.77 |
3.35 |
2143.9973 |
0.2237 |
54.60 |
||||
2230.5615 |
0.2328 |
52.77 |
Table 3: Depletion of the Lysine Peptide
Lysine Peptide |
||||||
Sample |
Peak Area at 220 nm |
Peptide Concentration [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1861.8433 |
0.2304 |
55.78 |
54.93 |
0.78 |
1.42 |
1926.2322 |
0.2384 |
54.25 |
||||
1904.2300 |
0.2357 |
54.77 |
||||
Test Item |
n.a.* |
|
|
|
|
|
n.a.* |
|
|
||||
n.a.* |
|
|
* not applicable due to co-elution
Table 4: 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 5: 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 6: Categorization of the Test Item
Prediction Model |
Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Item Ratio: 1:10 and 1:50) |
Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10) |
||||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
n.a. |
n.a. |
n.a. |
52.81 |
Moderate Reactivity |
sensitizer |
Positive Control |
64.92 |
High Reactivity |
sensitizer |
74.90 |
Moderate Reactivity |
sensitizer |
No alerts were matched. Endpoints not firing any alerts at the selected reasoning level were occupational asthma, respiratory sensitisation, photoallergenicity and skin sensitisation.
Table 1: Results of the Cytotoxicity Measurement
|
Concentration [µM] |
Cell Viability [%] |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
||
Solvent Control |
- |
100.0 |
100.0 |
100.0 |
0.0 |
Positive Control |
4.00 |
119.3 |
108.4 |
113.9 |
7.7 |
8.00 |
105.9 |
120.5 |
113.2 |
10.3 |
|
16.00 |
112.8 |
120.6 |
116.7 |
5.5 |
|
32.00 |
121.9 |
126.1 |
124.0 |
3.0 |
|
64.00 |
128.7 |
133.3 |
131.0 |
3.2 |
|
Test Item |
0.98 |
92.1 |
92.4 |
92.3 |
0.2 |
1.95 |
79.0 |
84.4 |
81.7 |
3.9 |
|
3.91 |
78.7 |
85.2 |
81.9 |
4.6 |
|
7.81 |
79.2 |
85.8 |
82.5 |
4.7 |
|
15.63 |
79.1 |
84.9 |
82.0 |
4.1 |
|
31.25 |
79.0 |
88.4 |
83.7 |
6.6 |
|
62.50 |
78.6 |
86.6 |
82.6 |
5.7 |
|
125.00 |
77.8 |
79.0 |
78.4 |
0.9 |
|
250.00 |
80.2 |
86.0 |
83.1 |
4.1 |
|
500.00 |
77.8 |
71.9 |
74.9 |
4.2 |
|
1000.00 |
78.7 |
73.3 |
76.0 |
3.8 |
|
2000.00 |
79.6 |
66.0 |
72.8 |
9.6 |
Table 2: Induction of Luciferase Activity Experiment 1
Experiment 1 |
Concentration [µM] |
Fold Induction |
Significance |
||||
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.05 |
1.50 |
1.19 |
1.25 |
0.23 |
|
8.00 |
1.38 |
1.20 |
1.30 |
1.29 |
0.09 |
|
|
16.00 |
1.33 |
1.21 |
1.27 |
1.27 |
0.06 |
|
|
32.00 |
1.43 |
1.52 |
1.32 |
1.42 |
0.10 |
|
|
64.00 |
4.85 |
3.80 |
4.18 |
4.28 |
0.53 |
* |
|
Test Item |
0.98 |
1.23 |
0.80 |
1.07 |
1.03 |
0.22 |
|
1.95 |
1.01 |
0.63 |
0.90 |
0.85 |
0.19 |
|
|
3.91 |
0.97 |
0.69 |
0.98 |
0.88 |
0.16 |
|
|
7.81 |
0.87 |
0.68 |
0.97 |
0.84 |
0.15 |
|
|
15.63 |
0.78 |
0.71 |
0.97 |
0.82 |
0.13 |
|
|
31.25 |
0.85 |
0.68 |
0.96 |
0.83 |
0.14 |
|
|
62.50 |
0.87 |
0.71 |
0.99 |
0.86 |
0.14 |
|
|
125.00 |
1.10 |
0.70 |
0.95 |
0.91 |
0.20 |
|
|
250.00 |
1.01 |
0.81 |
1.25 |
1.02 |
0.22 |
|
|
500.00 |
1.01 |
1.25 |
1.53 |
1.26 |
0.26 |
|
|
1000.00 |
1.08 |
1.78 |
2.08 |
1.64 |
0.51 |
|
|
2000.00 |
4.29 |
3.24 |
3.74 |
3.76 |
0.53 |
* |
* = significant induction according to Student’s t-test, p<0.05
Table 3: Induction of Luciferase Activity Experiment 2
Experiment 2 |
Concentration [µM] |
Fold Induction |
Significance |
||||
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.12 |
1.19 |
1.15 |
1.15 |
0.04 |
|
8.00 |
1.10 |
1.24 |
1.27 |
1.21 |
0.09 |
|
|
16.00 |
1.75 |
1.33 |
1.53 |
1.54 |
0.21 |
* |
|
32.00 |
1.87 |
1.70 |
1.96 |
1.85 |
0.13 |
* |
|
64.00 |
2.31 |
2.72 |
2.92 |
2.65 |
0.31 |
* |
|
Test Item |
0.98 |
1.15 |
1.11 |
1.18 |
1.15 |
0.04 |
|
1.95 |
1.12 |
1.15 |
1.07 |
1.12 |
0.04 |
|
|
3.91 |
1.04 |
1.05 |
1.14 |
1.08 |
0.06 |
|
|
7.81 |
1.20 |
1.03 |
1.07 |
1.10 |
0.09 |
|
|
15.63 |
1.06 |
1.07 |
1.38 |
1.17 |
0.18 |
|
|
31.25 |
1.12 |
1.06 |
1.14 |
1.11 |
0.04 |
|
|
62.50 |
1.22 |
1.13 |
1.19 |
1.18 |
0.04 |
|
|
125.00 |
1.33 |
1.08 |
1.22 |
1.21 |
0.12 |
|
|
250.00 |
1.42 |
1.46 |
1.58 |
1.48 |
0.08 |
|
|
500.00 |
1.71 |
1.81 |
1.57 |
1.70 |
0.12 |
* |
|
1000.00 |
2.03 |
1.78 |
1.72 |
1.84 |
0.17 |
* |
|
2000.00 |
2.99 |
2.43 |
2.83 |
2.75 |
0.29 |
* |
* = significant induction according to Student’s t-test, p<0.05
Table 4: Induction of Luciferase Activity – Overall Induction
|
Concentration [µM] |
Fold Induction |
Significance |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
|||
Solvent Control |
- |
1.00 |
1.00 |
1.00 |
0.00 |
|
Positive Control |
4.00 |
1.25 |
1.15 |
1.20 |
0.06 |
|
8.00 |
1.29 |
1.21 |
1.25 |
0.06 |
|
|
16.00 |
1.27 |
1.54 |
1.40 |
0.19 |
|
|
32.00 |
1.42 |
1.85 |
1.63 |
0.30 |
|
|
64.00 |
4.28 |
2.65 |
3.46 |
1.15 |
|
|
Test Item |
0.98 |
1.03 |
1.15 |
1.09 |
0.08 |
|
1.95 |
0.85 |
1.12 |
0.98 |
0.19 |
|
|
3.91 |
0.88 |
1.08 |
0.98 |
0.14 |
|
|
7.81 |
0.84 |
1.10 |
0.97 |
0.18 |
|
|
15.63 |
0.82 |
1.17 |
0.99 |
0.25 |
|
|
31.25 |
0.83 |
1.11 |
0.97 |
0.19 |
|
|
62.50 |
0.86 |
1.18 |
1.02 |
0.23 |
|
|
125.00 |
0.91 |
1.21 |
1.06 |
0.21 |
|
|
250.00 |
1.02 |
1.48 |
1.25 |
0.33 |
|
|
500.00 |
1.26 |
1.70 |
1.48 |
0.31 |
|
|
1000.00 |
1.64 |
1.84 |
1.74 |
0.14 |
* |
|
2000.00 |
3.76 |
2.75 |
3.25 |
0.71 |
* |
* = significant induction according to Student’s t-test, p<0.05
Table 5: Additional Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Mean |
SD |
EC1.5[µM] |
810.73 |
268.21 |
539.47 |
383.62 |
Imax |
3.76 |
2.75 |
3.25 |
0.71 |
IC30[µM] |
n.a. |
1455.58 |
1455.58 |
- |
IC50[µM] |
n.a. |
n.a. |
- |
- |
n.a. = not applicable
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
Sensitisation
The skin sensitizing potential of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-thiazolium (CAS 10023-48-0) was evaluated in a combination of non-animal methods (in silico, in chemico and in vitro).
(Q)SAR
The skin sensitisation potential of the test substance was predicted using a QSAR model Derek Nexus (5.0.1) and Nexus (2.1.0). A prediction of the potential for skin sensitisation, respiratory sensitisation, occupational asthma and photoallergenicity was performed. A prediction for a specific hazard potential is given as an alert, which consists of a toxicophore (a substructure known or thought to be responsible for the toxicity) and is associated with literature references, comments and examples. There were no alerts for any of the predictions performed.
In chemico/in vitro
The skin sensitising potential of the test substance was assessed in a Direct Peptide Reactivity Assay performed according to OECD guideline 442C and in compliance with GLP (reference 7.4.1-1). 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. The mean depletion of the cysteine peptide was 52.81%. Using the prediction model defined in the guideline, the test substance is allocated to the reactivity class “moderate reactivity”. In the lysine run a co-elution of the test substance with the lysine peptide peak was observed. However, all validity criteria were fulfilled, and this shift was not considered having an influence on the quality or validity of the overall results. The positive control showed high reactivity towards the synthetic peptides as the mean depletion of both peptides was 64.92%.
Under the conditions of the test, the test substance showed reactivity towards the cysteine peptide. 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 is required.
The activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test in the transgenic KeratinoSens™ cell line performed according to OECD guideline 442D and in compliance with GLP (reference 7.4.1-2). Cells were incubated with test substance concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000, 2000 µM for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. The study was conducted in two independent experiments. In the first experiment a max luciferase activity (Imax) induction of 3.76 was determined at a test substance concentration of 2000 µM. The corresponding cell viability was 79.6%. In the second experiment a max luciferase activity (Imax) induction of 2.75 was determined at a test substance concentration of 2000 µM. The corresponding cell viability was 66.0%. Therefore in both experiments the luciferase activity induction was >1.5. The lowest tested concentration with a significant luciferase induction >1.5 (1.70) was found to be 500 µM. The corresponding cell viability was >70% (71.9%). The EC1.5 was 810.73 µM and 268.21 µM in Experiment 1 and 2, respectively. Under the conditions of the test, the test substance has a keratinocyte activating potential.
Conclusion
A weight of evidence approach was applied to the available data on skins sensitisation, as the result of the QSAR prediction was negative, while the results of the 2 in chemico/in vitro tests were positive. The positive results of validated in chemico/in vitro tests covering 2 key events in the molecular initiating event leading to skin sensitisation are considered more reliable than the result of a QSAR prediction, which is based on (mainly structural) similarities between the test substance and known skin sensitisers. Therefore the test substance is considered to show a skin sensitising potential.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
The available data on skin sensitisation of the test substance meet the classification criteria for Skin Sens 1, H317 according to Regulation (EC) 1272/2008.
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