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EC number: 231-007-9 | CAS number: 7403-42-1
- 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
Skin sensitisation (OECD 442C and 442D): negative
Skin sensitisation (OECD 442E): positive
WoE conclusion from in vitro skin sensitisation battery: not 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:
- 06 - 13 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:
- 04 Feb 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Medicines & Healthcare products Regulatory Agency, UK
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- 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 UV detection at 220 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.
TEST SYSTEM
- Supplier: AnaSpec
- Synthetic cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/L
Purity: 95%
Expiry date: 5 years
- Synthetic lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/L
Purity: 94%
Expiry date: 5 years
SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile
The solubility of the test item in acetonitrile was assessed at a concentration of 100 mM.
POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Batch number: MKBR2427V
- Purity: >95%
- Expiry date: Feb 2019
The positive control was prepared at a concentration of 100 mM.
STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.
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 OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance/positive control); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance/positive control)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h prior to initiation of the analysis run
NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- 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
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% B: 10, 25, 90, 90, 10, 10
- Detector Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.0167, 0.0334, 0.0667, 0.133, 0.267 and 0.534 mM
- Column temperature: 30 °C - Key result
- Run / experiment:
- other: ≥ 22 h incubation
- Parameter:
- other: % depletion of cysteine-containing peptide
- Remarks:
- (mean value of 3 replicates)
- Value:
- 0.703
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: ≥ 22 h incubation
- Parameter:
- other: % depletion of lysine-containing peptide
- Remarks:
- (mean value of 3 replicates)
- Value:
- -0.411
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Run / experiment:
- other: ≥ 22 h incubation
- Parameter:
- other: % overall mean depletion
- Remarks:
- (negative numbers count as zero)
- Value:
- 0.352
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay. The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.
ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met. - Interpretation of results:
- other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C
- Conclusions:
- Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.
- Executive summary:
Protein reactivity of the test substance was determined by a Direct Peptide Reactivity Assay according to OECD 442C and in compliance with GLP (2017).The overall mean percent cysteine and lysine depletion is 0.352 and therefore the test substance is allocated to the “no or minimal reactivity” class (mean % depletion > 0 ≤ 6.38). Thus, the DPRA prediction for protein reactivity is considered negative.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 08 Mar - 12 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 04 Feb 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, 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 to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.
TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 8 (Experiment 1), 6 (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-glucose 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, 62.5, 125, 250, 500, 1000 and 2000 µM
CONTROLS
Solvent/ Negative control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1% (v/v)
Positive control
- Substance: cinnamic aldehyde
- Final concentration: 4 - 64 µM
EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h
NUMBER OF REPLICATIONS: triplicates in two independent experiments (treatment group, positive control); sextuplicates in two independent experiments (solvent control)
DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 600 nm
DETERMINATION OF LUMINESCENCE
- Cell lysis reagent: Luciferase Cell Culture Lysis 5x Reagent Kit (Promega, Cat. No. E1531, Lot No. 0000087405)
- Luciferase reagent: Luciferase Assay Kit (Promega, Cat. no. E1501, Lot No. 0000237533)
- Device: plate reader - Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: maximum luciferase activity (Imax)
- Value:
- 1.06
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: test item concentration: 3.91 µM; cell viability: 104.5%
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: maximum luciferase activity (Imax)
- Value:
- 0.94
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: test item concentration: 3.91 and 1000 µM; cell viability: 104.5 and 99.4%, respectively
- Other effects / acceptance of results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY: No evidence of cytotoxicity (cell viability < 70%) was observed.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The average coefficient of variation of the luminescence reading for the vehicle control was < 20% (11.7% and 16.4% 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 (2.44 and 3.90 in Experiment 1 and 2, respectively) and the calculated EC1.5 value was between 7 and 34 µM (21.80 and 21.42 µM in Experiment 1 and 2, respectively). - Interpretation of results:
- other: activation of keratinocytes negative according to OECD 442D
- Conclusions:
- Under the conditions of the ARE-Nrf2 Luciferase test the test substance did not induce luciferase activity in the transgenic KeratinoSens™ cell line.
- Executive summary:
In a second study, the activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test (LuSens) in the transgenic KeratinoSens™ cell line according to OECD Guideline 442D and in compliance with GLP (2017). In two independent experiments, a max luciferase activity induction (Imax) of 1.06 (at 3.91 µM) and 0.94 (at 3.91 and 1000 µM) was determined, respectively. The corresponding cell viabilities were 98.4% and 110.5% (99.2%), respectively.Therefore, in this study the test substance did not induce luciferase activity in the transgenic KeratinoSens™ cell line.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 27 Mar - 13 Apr 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test)
- Version / remarks:
- (Jul 2016)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of study:
- activation of dendritic cells
- Details on the study design:
- TEST CELL LINE
- Strain: THP-1 cells (human monocytic leukemia cell line)
- Source: American Type Culture Collection
- Passage number: 24 and 25 (XTT test); 12 and 13 (h-CLAT)
CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% (v/v) FBS, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) pyruvate and 1% (v/v) L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5.0 ± 0.5
DOSE FINDING ASSAY BY CYTOTOXICITY MEASUREMENT (XTT TEST)
The doses investigated in the main experiment (h-CLAT) were determined with two XTT tests. The XTT test is a method to investigate the cytotoxicity of a test substance. The test is based on the cleavage of the yellow tetrazolium salt, sodium 3'-(1-phenylaminocarbonyl)-(3,4-tetrazolium)-bis-(4–methoxy-6-nitro)-benzenesulfonic acid hydrate (XTT), to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable concentration showing 75% cell viability (CV75).
CONTROLS
Negative Control
- Substance: culture medium
Solvent control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2%
EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h
TEST CONCENTRATIONS
- Justification for top dose: The test substance was soluble in DMSO up to and including 5000 µg/mL as tested by a solubility test.
39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL
NUMBER OF REPLICATIONS: 7 replicates per concentration in two independent experiments
MEASUREMENT
- Device: microplate reader (Versamax Molecular Devices)
- Wavelength: 450 nm
- Reference wavelength: 690 nm
EVALUATION CRITERIA
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance as compared to the solvent control is calculated using the formula:
Relative absorbance = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance solvent control) - (absorbance blank)]
To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the vehicle control (CV75) the following formula is used:
CV75 = Conc. > 75 - ([(Conc. > 75) - (Conc. < 75)] x [(% > 75) - 75] / [(% > 75) - (% < 75)])
a) Conc. > 75: max. measured concentration with the % of solvent control > 75%
b) Conc. < 75: min. measured concentration with the % of solvent control < 75%
c) % > 75: relative absorbance at a) in %
d) % < 75: relative absorbance at b) in %
ACCEPTANCE CRITERIA
The XTT test is considered to be acceptable if it meets the following criteria:
- mean absorbance of the negative control is ≥ 0.5
- mean viability of the solvent control is ≥ 90% compared to the negative (medium) control
h-CLAT (main study)
The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared to vehicle control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.
CONTROLS
Negative control
- Substance: culture medium
Solvent control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene (DNCB) in DMSO
- Concentration: 2 and 3 µg/mL
EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h
TEST CONCENTRATIONS
- Justification for top dose: The highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75.
79, 95, 114, 137, 164, 197, 236 and 284 μg/mL
NUMBER OF REPLICATIONS: triplicates for the different stainings in two independent experiments
STAINING
- Antibodies: FITC anti-CD86 (Clone: Fun-1); FITC anti-CD54 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C
- Duration: 30 ± 5 min
MEASUREMENT
- Device: flow cytometer FACSCalibur (Becton Dickinson)
EVALUATION CRITERIA
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration:
RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance treated isotype control cells) / (MFI of solvent control cells) - (MFI of solvent isotype control cells)] x 100
MFI: geometric mean fluorescence intensity (GeoMean)
The cell viability is calculated as follows for each concentration:
Cell viability (%) = (mean cytotox of solvent control cells) / (mean cytotox of test substance treated cells) x 100
Where the mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean (7-AAD) CD54 and GeoMean (7-AAD) CD86.
The test substance is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.
ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of negative control is adjusted to 100% and the cell viability of the solvent control should be more than 90% in comparison to the negative control.
- In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For negative and solvent controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- The cell viability of at least 4 doses in each experiment should be ≥ 50%.
- For the test substance resulting in negative outcome, the cell viability at the 1.2 × CV75 value should be less than 90%. If the cell viability at the 1.2 × CV75 value is more than 90% for a positive tested test substance, the data should be discarded. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test substance are accepted independent by the cell viability. - Key result
- Run / experiment:
- other: 24 h incubation
- Parameter:
- other: relative fluorescence intensity of CD54 (%)
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: in 2 independent runs
- Key result
- Run / experiment:
- other: 24 h incubation
- Parameter:
- other: relative fluorescence intensity of CD86
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: in 2 independent runs
- Other effects / acceptance of results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
Cytotoxic effects (threshold of cytotoxicity: cell viability < 75%) were observed following incubation with the test item starting with the concentration of 312.5 μg/mL up to the highest tested concentration (5000 μg/mL) in both XTT tests. The mean CV75 value of both XTT tests was calculated to be 236.45 μg/mL.
h-CLAT
The cell viability for the test substance was > 50% in all tested concentrations of both independent experiments.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: In the solvent control, RFI values compared to the negative control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 exceeded the positive criteria (CD86 >150% and CD54 >200%) and the cell viability was > 50%. - Interpretation of results:
- other: activation of dendritic cells positive according to OECD 442E
- Conclusions:
- Under the conditions of the Human Cell Line Activation Test the test substance increased the expression of both cell surface markers, CD54 and CD86, which is associated with the process of activation of monocytes and dendritic cells. Based on this result, the h-CLAT prediction is considered positive for skin sensitisation.
- Executive summary:
Dendritic cell response of the test substance was investigated by a Human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E and in compliance with GLP (2017). The test substance increased the expression of cell suface marker CD54 and CD86 to greater than 200% and 150%, respectively in two independent experiments. Thus, the h-CLAT prediction for activation of dendritic cells is considered positive.
Referenceopen allclose all
Table 5. Mean peptide depletion of cysteine-containing peptide.
|
Peak area (µV.sec) |
Peptide concentration1 (µg/mL) |
Peptide depletion2(%) |
Mean depletion ± SD (%) |
Positive control |
245051 |
104.38 |
72.2 |
72.3 ± 0.10 |
243414 |
103.68 |
72.4 |
||
244930 |
104.33 |
72.2 |
||
Test substance |
880480 |
378.50 |
0.152 |
0.703 ± 0.20 |
870766 |
374.31 |
1.25 |
||
9672253 |
- |
- |
SD: Standard Deviation
1 Samples prepared at a concentration of 376 µg/mL (0.5 mM)
2 Calculated against a mean Reference Control area of 881820 µV.sec(n=6)
3 Result not reported due to loss of solvent during incubation caused by a loose vial cap.
Table 6. Mean peptide depletion of lysine-containing peptide.
|
Peak area (µV.sec) |
Peptide concentration1 (µg/mL) |
Peptide depletion2(%) |
Mean depletion ± SD (%) |
Positive control |
311319 |
161.29 |
58.9 |
58.4± 0.41 |
315241 |
163.33 |
58.3 |
||
317420 |
164.47 |
58.1 |
||
Test substance |
759314 |
394.88 |
-0.343 |
-0.411 ± 0.16 |
758928 |
394.67 |
-0.292 |
||
761248 |
395.88 |
-0.598 |
SD: Standard Deviation
1 Samples prepared at a concentration of 388 µg/mL (0.5 mM)
2 Calculated against a mean Reference Control area of 756720 µV.sec (n=6)
Table 2. Results of the cytotoxicity measurement
|
Concentration [µM] |
Cell viability [%] |
|||
|
|
Experiment 1 |
Experiment 2 |
Mean |
SD |
Solvent control |
- |
100 |
100 |
100 |
0.0 |
Positive control |
4 |
107.0 |
105.4 |
106.2 |
1.2 |
8 |
101.3 |
98.4 |
99.8 |
2.0 |
|
16 |
100.3 |
93.8 |
97.0 |
4.6 |
|
32 |
112.3 |
87.2 |
99.7 |
17.7 |
|
64 |
136.6 |
86.5 |
111.5 |
35.4 |
|
Test substance |
0.98 |
117.2 |
104.8 |
111.0 |
8.7 |
1.95 |
108.5 |
110.8 |
109.7 |
1.6 |
|
3.91 |
98.4 |
110.5 |
104.5 |
8.6 |
|
7.81 |
100.0 |
111.4 |
105.7 |
8.1 |
|
15.63 |
99.0 |
106.2 |
102.6 |
5.1 |
|
31.25 |
90.5 |
106.6 |
98.5 |
11.4 |
|
62.50 |
108.0 |
102.6 |
105.3 |
3.8 |
|
125 |
97.6 |
112.4 |
105.0 |
10.4 |
|
250 |
93.1 |
104.5 |
98.8 |
8.1 |
|
500 |
103.7 |
104.2 |
104.0 |
0.4 |
|
1000 |
99.6 |
99.2 |
99.4 |
0.2 |
|
2000 |
95.8 |
98.8 |
97.3 |
2.2 |
Table 3. Induction of luciferase activity
|
Concentration [µM] |
Experiment 1 |
Experiment 2 |
||
|
|
Mean fold induction |
SD |
Mean fold induction |
SD |
Solvent control |
- |
1.00 |
0.00 |
1.00 |
0.00 |
Positive control |
4 |
1.11 |
0.07 |
1.06 |
0.03 |
8 |
1.09 |
0.08 |
1.11 |
0.13 |
|
16 |
1.39 |
0.22 |
1.23 |
0.09 |
|
32 |
1.70* |
0.26 |
2.02* |
0.31 |
|
64 |
2.44* |
0.33 |
3.90* |
0.19 |
|
Test substance |
0.98 |
1.00 |
0.09 |
0.89 |
0.24 |
1.95 |
1.04 |
0.05 |
0.84 |
0.28 |
|
3.91 |
1.06 |
0.07 |
0.94 |
0.20 |
|
7.81 |
0.96 |
0.07 |
0.86 |
0.18 |
|
15.63 |
0.98 |
0.13 |
0.92 |
0.26 |
|
31.25 |
0.97 |
0.11 |
0.89 |
0.24 |
|
62.50 |
1.00 |
0.03 |
0.90 |
0.31 |
|
125 |
0.97 |
0.06 |
0.88 |
0.22 |
|
250 |
0.95 |
0.10 |
0.82 |
0.12 |
|
500 |
0.93 |
0.09 |
0.87 |
0.25 |
|
1000 |
1.01 |
0.07 |
0.94 |
0.16 |
|
2000 |
0.94 |
0.28 |
0.91 |
0.17 |
*=significant induction according to Student's t-test,p<0.05
Table 1: Results of the first XTT Test
|
Concentration (µg/mL) |
Mean absorbance* |
SD |
Blank |
Absorbance in % of vehicle control** |
Negative control (medium) |
- |
0.620 |
0.029 |
0.228 |
110.52 |
Solvent Control (DMSO) |
- |
0.583 |
0.028 |
0.229 |
100.0 |
Test substance |
39.1 |
0.534 |
0.011 |
0.223 |
87.91 |
78.1 |
0.587 |
0.024 |
0.219 |
103.76 |
|
156.3 |
0.548 |
0.017 |
0.222 |
92.06 |
|
312.5 |
0.470 |
0.026 |
0.218 |
71.25 |
|
625 |
0.418 |
0.015 |
0.224 |
54.72 |
|
1250 |
0.233 |
0.009 |
0.228 |
1.59 |
|
2500 |
0.239 |
0.010 |
0.236 |
0.85 |
|
5000p |
0.242 |
0.004 |
0.237 |
1.53 |
p: precipitation
*: mean absorbance (absolute) of 7 wells
**: relative absorbance
Table 2: Results of the second XTT Test
|
Concentration (µg/mL) |
Mean absorbance* |
SD |
Blank |
Absorbance in % of vehicle control** |
Negative control (medium) |
- |
0.678 |
0.033 |
0.220 |
106.64 |
Solvent Control (DMSO) |
- |
0.664 |
0.045 |
0.235 |
100.0 |
Test substance |
39.1 |
0.736 |
0.066 |
0.225 |
118.92 |
78.1 |
0.631 |
0.045 |
0.233 |
92.72 |
|
156.3 |
0.573 |
0.032 |
0.234 |
78.98 |
|
312.5 |
0.492 |
0.024 |
0.236 |
59.69 |
|
625 |
0.425 |
0.022 |
0.238 |
43.48 |
|
1250 |
0.254 |
0.007 |
0.243 |
2.60 |
|
2500 |
0.248 |
0.004 |
0.231 |
3.94 |
|
5000p |
0.246 |
0.004 |
0.221 |
5.71 |
p: precipitate
*: mean absorbance (absolute) of 7 wells
**: relative absorbance
The CV75 value of the first XTT test: 284.4μg/mL
The CV75 value of the second XTT test: 188.5μg/mL
The mean CV75 value of both XTT tests: 236.45μg/mL
Table 3. Results of the first h-CLAT run
|
Concentration (µg/mL) |
RFI (%) CD54 Antibody |
RFI (%) CD86 Antibody |
Cell Viability (%) |
Negative control (medium) |
- |
100.0 |
100.0 |
100.0 |
Solvent control (DMSO) |
- |
100.0 |
100.0 |
100.0 |
Positive control (DNCB) |
2.0 |
435.4* |
692.4* |
62.2 |
3.0 |
654.9* |
738.9* |
63.0 |
|
Test substance |
79 |
126.1 |
80.1 |
95.3 |
95 |
163.8 |
127.0 |
87.8 |
|
114 |
179.7 |
127.0 |
86.7 |
|
137 |
198.6 |
107.8 |
83.8 |
|
164 |
189.9 |
95.0 |
79.9 |
|
197 |
218.8* |
118.4 |
79.3 |
|
236 |
243.5* |
122.7 |
71.0 |
|
284 |
265.2* |
158.2* |
72.3 |
* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).
Table 4. Results of the second h-CLAT run
|
Concentration (µg/mL) |
RFI (%) CD54 Antibody |
RFI (%) CD86 Antibody |
Cell Viability (%) |
Negative control (medium) |
- |
100.0 |
100.0 |
100.0 |
Solvent control (DMSO) |
- |
100.0 |
100.0 |
100.0 |
Positive control (DNCB) |
2.0 |
447.4* |
728.2* |
62.5 |
3.0 |
350.5* |
464.1* |
65.0 |
|
Test substance |
79 |
158.9* |
92.8 |
90.3 |
95 |
171.1 |
109.8 |
92.1 |
|
114 |
200.0* |
116.3 |
89.8 |
|
137 |
216.7* |
122.9 |
91.4 |
|
164 |
222.2* |
127.5 |
86.6 |
|
197 |
242.2* |
143.1 |
80.0 |
|
236 |
246.7* |
200.0* |
66.5 |
|
284 |
298.9* |
364.1* |
52.3 |
* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).
Endpoint conclusion
- Additional information:
The skin sensitizing potential of the test substance was evaluated in a combination of non-animal methods (in chemico and in vitro). Three key events of skin sensitisation a) molecular interaction with skin proteins, b) inflammatory response in keratinocytes and c) activation of dendritic cells were addressed.
a) Molecular interaction with skin proteins
Protein reactivity of the test substance was determined by a Direct Peptide Reactivity Assay according to OECD 442C and in compliance with GLP (2017).The overall mean percent cysteine and lysine depletion is 0.352 and therefore the test substance is allocated to the “no or minimal reactivity” class (mean % depletion > 0 ≤ 6.38). Thus, the DPRA prediction for protein reactivity is considered negative.
b) Activation of keratinocytes
In a second study, the activation of keratinocytes of the test substance was investigated in an ARE-Nrf2 Luciferase Test (LuSens) in the transgenic KeratinoSens™ cell line according to OECD Guideline 442D and in compliance with GLP (2017). In two independent experiments, a max luciferase activity induction (Imax) of 1.06 (at 3.91 µM) and 0.94 (at 3.91 and 1000 µM) was determined, respectively. The corresponding cell viabilities were 98.4% and 110.5% (99.2%), respectively. Therefore, in this study the test substance did not induce luciferase activity in the transgenic KeratinoSens™ cell line.
c) Activation of dendritic cells
In a third study, dendritic cell response of the test substance was investigated by a Human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E and in compliance with GLP (2017). The test substance increased the expression of cell suface marker CD54 and CD86 to greater than 200% and 150%, respectively in two independent experiments. Thus, the h-CLAT prediction for activation of dendritic cells is considered positive.
Conclusion
Based on a weight of evidence approach considering two negative and one positive results in the in vitro/in chemico test battery, the test substance is not considered to be a skin sensitiser.
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 do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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