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EC number: 937-960-3 | 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
Endpoint summary
Administrative data
Description of key information
Skin sensitisation (OECD 442C and 442D): negative
Skin sensitisation (OECD 442E): positive
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:
- 9 - 19 Mar 2016
- 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:
- 2015
- Deviations:
- yes
- Remarks:
- no information on vehicle control and reference substances provided
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- TEST SYSTEM
- Supplier: AnaSpec
- Cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/mol
Purity: 95%
Expiry date: 5 years
- Lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/mol
Purity: 90 - 95%
Expiry date: 5 years
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.
VEHICLE CONTROL
- Substance: acetonitrile
POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Supplier: SAFC
- Batch number: MKBR2427V
- Purity: >95%
- Expiry date: Feb 2019
STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.
POSITIVE CONTROL PREPARATION
The positive control was prepared at a concentration of 100 mM.
TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile.
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
- Peptide ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h
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
- Wavelength: 220 nm
- Injection volume: 2 µL
- 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:
- -2.48
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not specified
- 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:
- 5.85
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Run / experiment:
- other: ≥ 22 h incubation
- Parameter:
- other: % overall mean depletion
- Value:
- 1.69
- Vehicle controls validity:
- not specified
- Negative controls validity:
- not specified
- 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)
- Remarks:
- according to OECD TG 442C
- Conclusions:
- Under the conditions of the Direct Peptide Reactivity Assay the test substance showed no or minimal peptide reactivity.
- Executive summary:
Solutions of test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result places the test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 5 Sep - 14 Oct 2016
- 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 4 February 2015
- Deviations:
- no
- GLP compliance:
- yes
- 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: 12 (Experiment 1), 14 (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, 61.5, 125, 250, 500, 1000 and 2500 µM
CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1%
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
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
- Luciferase reagent: Luciferase Assay Kit (Promega, Cat. no. E1501, Lot. no. 0000206987)
- Device: plate reader - Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: maximum luciferase activity induction
- Value:
- 1.14
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: maximum luciferase activity induction
- Value:
- 1.14
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY: Clear evidence of cytotoxicity (cell viability < 70%) was observed at 500 µM and above.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The average coefficient of variation of the luminescence reading for the vehicle control was < 20% (10.5% 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 (2.12 and 2.88 in Experiment 1 and 2, respectively) and the calculated EC1.5 value was between 7 and 30 µM (27.06 and 21.54 µM in Experiment 1 and 2, respectively). - Interpretation of results:
- other: negative
- Remarks:
- according to OECD TG 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 the first experiment no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.14 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 111.6%. Therefore, no EC1.5could be calculated.
In the second experiment no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.14 was determined at a test item concentration of 0.98 µM. The corresponding cell viability was 141.3%. Therefore, no EC1.5could be calculated.
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as non sensitiser.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 10 Mar - 13 Apr 2016
- 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:
- December 2015
- Deviations:
- yes
- Remarks:
- cytotoxicity measurement and estimation of the CV75 value was performed by XTT test instead of flow cytometry
- 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
- Source: American Type Culture Collection
- Passage number: 6 - 7 (XTT test); 14 - 16 (h-CLAT)
CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) pyruvate and 1% (v/v) L-glutamine
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5.0 ± 0.5
The following information relate to details on XTT
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 and on different days to obtain a reliable concentration showing 75% cell viability (CV75).
CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2 - 0.5%
EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h
TEST CONCENTRATIONS
- Justification for top dose: The test substance was soluble in DMSO up to and including 250 µg/mL.
1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125 and 250 µg/mL
NUMBER OF REPLICATIONS: 4 samples per dose groups were tested 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 vehicle 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 vehicle control > 75%
b) Conc. < 75: min. measured concentration with the % of vehicle 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 vehicle control is ≥ 70%
The following information relate to details on h-CLAT
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 solvent control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.
CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: DMSO
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene in DMSO
- Concentration: 2 and 3 µg/mL
EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h
TEST CONCENTRATIONS
- Justification for top dose: The highest concentration used was 1.2 × mean CV75 of both XTT tests. The calculated mean CV75 value of both XTT tests was 182.8 μg/mL. However, due to a calculation error of the CV75 value, the highest test substance concentration used was 260.8 μg/mL instead of 219.4 μg/mL.
72.8, 87.3, 104.8, 125.8, 150.9, 181.1, 217.3 and 260.8 µg/mL
NUMBER OF REPLICATIONS: at least in triplicates for the different stainings in two independent experiments
STAINING
- Antibodies: FITC anti-CD54 (Clone: Fun-1); FITC anti-CD86 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C
- Duration: 30 ± 5 min
MEASUREMENT
- Device: 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 isotype control cells) / (MFI of vehicle control cells) - (MFI of vehicle 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 vehicle 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 vehicle control should be more than 90% in comparison to the negative control.
- In the positive control, RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- 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 vehicle controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- 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 will be acceptable. 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.
- The cell viability of at least 4 doses in each experiment should be ≥50%. - 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
- 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
- Other effects / acceptance of results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
Cytotoxic effects were observed following incubation with the highest tested concentration (250 µg/mL). The viability of the cells was reduced to 59.15 and 58.19%, respectively, in both XTT tests (threshold of cytotoxicity: < 75%). The calculated mean CV75 value of both XTT tests was 182.8 μg/mL. However, due to a calculation error of the CV75 value, the highest test substance concentration used in the main experiment was 260.8 μg/mL instead of 219.4 μg/mL.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: In the vehicle 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: positive
- Remarks:
- according to OECD TG 442E
- Conclusions:
- Under the conditions of the Human Cell Line Activation Test the test substance increased the expression of CD86, a cell surface marker 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.
Referenceopen allclose all
Table 5. Mean peptide depletion of cysteine-containing peptide.
|
Peak area (µV.sec) |
Peptide concentration [µg/mL)* |
Peptide depletion (%)** |
Mean depletion ± CV (%) |
Test substance |
964704 |
389 |
-2.63 |
-2.48 ± 1.41 |
949103 |
382 |
-0.968 |
||
976123 |
394 |
-3.84 |
||
Positive control |
234824 |
96.3 |
75.0 |
74.0 ± 3.77 |
243906 |
100 |
74.1 |
||
253197 |
104 |
73.1 |
*: Samples prepared at a concentration of 376 μg/mL (0.5 mM)
**: Calculated against a mean reference control peak area of 940000 μV.sec (n=6)
Table 6. Mean peptide depletion of lysine-containing peptide.
|
Peak area (µV.sec) |
Peptide concentration [µg/mL)* |
Peptide depletion (%)** |
Mean depletion ± CV (%) |
Test substance |
743844 |
365 |
5.69 |
5.85 ± 0.50 |
745400 |
366 |
5.49 |
||
738354 |
362 |
6.38 |
||
Positive control |
341222 |
167 |
56.7 |
55.8 ± 7.06 |
376020 |
184 |
52.3 |
||
328441 |
161 |
58.4 |
*: Samples prepared at a concentration of 388 μg/mL (0.5 mM)
**: Calculated against a mean reference control peak area of 788680 μV.sec (n=6)
Table1. Results of the cytotoxicity measurement
|
Concentration [µM] |
Cell Viability (%) |
||
Experiment 1 |
Experiment 2 |
Mean ± SD |
||
Vehicle control |
- |
100 |
100 |
100 ± 0.0 |
Positive control |
4 |
98.8 |
96.0 |
97.4 ± 2.0 |
8 |
109.0 |
104.4 |
106.7 ± 3.3 |
|
16 |
110.2 |
108.5 |
109.3 ± 1.2 |
|
32 |
111.3 |
115.2 |
113.2 ± 2.7 |
|
64 |
125.1 |
125.9 |
125.5 ± 0.6 |
|
Test substance |
0.98 |
112.9 |
141.3 |
127.1 ± 20.1 |
1.95 |
98.5 |
112.8 |
105.7 ± 10.2 |
|
3.91 |
106.8 |
106.3 |
106.5 ± 0.3 |
|
7.81 |
115.9 |
101.9 |
108.9 ± 9.9 |
|
15.63 |
111.6 |
104.5 |
108.0 ± 5.0 |
|
31.25 |
124.5 |
102.6 |
113.6 ± 15.5 |
|
62.50 |
105.4 |
87.7 |
96.5 ± 12.5 |
|
125 |
95.2 |
89.5 |
92.3 ± 4.1 |
|
250 |
108.8 |
68.4 |
88.6 ± 28.6 |
|
500 |
-0.2 |
0.2 |
0.0 ± 0.3 |
|
1000 |
0.0 |
0.7 |
0.4 ± 0.5 |
|
2000 |
0.1 |
0.6 |
0.4 ± 0.4 |
Table 2. Induction of luciferase activity
|
Concentration [µM] |
Fold Induction |
||
Experiment 1 |
Experiment 2 |
Mean ± SD |
||
Solvent control |
- |
1.00 |
1.00 |
1.00 ± 0.00 |
Positive control |
4 |
1.10 |
1.16 |
1.13 ± 0.05 |
8 |
1.24 |
1.14 |
1.19 ± 0.07 |
|
16 |
1.29 |
1.31 |
1.30 ± 0.01 |
|
32 |
1.59 |
1.85 |
1.72 ± 0.18* |
|
64 |
2.12 |
2.88 |
2.50 ± 0.54* |
|
Test substance |
0.98 |
1.02 |
1.14 |
1.08 ± 0.08 |
1.95 |
1.07 |
1.03 |
1.05 ± 0.03 |
|
3.91 |
1.01 |
1.05 |
1.03 ± 0.03 |
|
7.81 |
1.00 |
1.00 |
1.00 ± 0.00 |
|
15.63 |
1.14 |
0.99 |
1.06 ± 0.11 |
|
31.25 |
1.01 |
1.05 |
1.03 ± 0.03 |
|
62.50 |
1.02 |
0.96 |
0.99 ± 0.04 |
|
125 |
1.05 |
1.11 |
1.08 ± 0.04 |
|
250 |
1.02 |
0.97 |
0.99 ± 0.03 |
|
500 |
- |
- |
- |
|
1000 |
- |
- |
- |
|
2000 |
- |
- |
- |
*: significant induction according to Student’s T-test, p<0.05
Table 1: Results of the first XTT test
|
Concentration [µg/mL] |
Mean absorbance* |
Blank |
Absorbance in % of vehicle control** |
Negative control |
- |
0.600 ± 0.028 |
0.229 |
93.68 |
Vehicle control |
- |
0.613 ± 0.040 |
0.217 |
100.0 |
Test substance |
1.95 |
0.635 ± 0.059 |
0.220 |
104.78 |
3.91 |
0.660 ± 0.041 |
0.224 |
109.88 |
|
7.81 |
0.656 ± 0.058 |
0.224 |
109.18 |
|
15.63 |
0.610 ± 0.029 |
0.225 |
97.45 |
|
31.25 |
0.599 ± 0.071 |
0.227 |
93.98 |
|
62.5 |
0.637 ± 0.081 |
0.234 |
101.81 |
|
125 |
0.577 ± 0.050 |
0.230 |
87.78 |
|
250 |
0.462 ± 0.035 |
0.228 |
59.15 |
*: mean absorbance (absolute) of 4 wells
**: relative absorbance
Table 2: Results of the second XTT test
|
Concentration [µg/mL] |
Mean absorbance* |
Blank |
Absorbance in % of vehicle control** |
Negative control |
- |
0.784 ± 0.023 |
0.262 |
103.84 |
Vehicle control |
- |
0.777 ± 0.040 |
0.274 |
100.00 |
Test substance |
1.95 |
0.794 ± 0.025 |
0.277 |
102.95 |
3.91 |
0.759 ± 0.015 |
0.276 |
95.96 |
|
7.81 |
0.776 ± 0.046 |
0.272 |
100.33 |
|
15.63 |
0.750 ± 0.037 |
0.266 |
96.36 |
|
31.25 |
0.784 ± 0.024 |
0.275 |
101.31 |
|
62.5 |
0.772 ± 0.021 |
0.277 |
98.34 |
|
125 |
0.728 ± 0.018 |
0.273 |
90.42 |
|
250 |
0.559 ± 0.025 |
0.267 |
58.19 |
*: mean absorbance (absolute) of 4 wells
**: relative absorbance
Table 3. Results of the first h-CLAT run
|
Concentration [µg/mL] |
Antibody / ISO |
MFI GeoMean (FITC) |
MFI - ISO |
Csto(Geo) GeoMean (7-AAD) |
Mean Cyto |
RFI (%) |
Viability (%) |
Negative control |
- |
ISO |
1.92 |
- |
1.83 |
1.7 |
- |
100.0 |
CD54 |
3.02 |
1.10 |
1.62 |
100.0 |
||||
CD86 |
4.41 |
2.49 |
1.64 |
100.0 |
||||
Vehicle control |
- |
ISO |
1.85 |
- |
2.10 |
1.8 |
- |
100.0 |
CD54 |
2.97 |
1.12 |
1.70 |
100.0 |
||||
CD86 |
3.89 |
2.04 |
1.46 |
100.0 |
||||
Positive control |
2 |
ISO |
2.31 |
- |
1.85 |
1.5 |
- |
113.9 |
CD54 |
9.87 |
7.56 |
1.44 |
675.0* |
||||
CD86 |
9.65 |
7.34 |
1.33 |
359.8* |
||||
3 |
ISO |
2.32 |
- |
2.44 |
1.8 |
- |
98.1 |
|
CD54 |
10.73 |
8.41 |
1.43 |
750.9* |
||||
CD86 |
10.24 |
7.92 |
1.49 |
388.2* |
||||
Test substance |
72.77 |
ISO |
1.95 |
- |
1.96 |
1.7 |
- |
104.2 |
CD54 |
3.37 |
1.42 |
1.51 |
126.8 |
||||
CD86 |
3.28 |
1.33 |
1.58 |
65.2 |
||||
87.33 |
ISO |
1.93 |
- |
1.83 |
1.7 |
- |
103.7 |
|
CD54 |
3.17 |
1.24 |
1.80 |
110.7 |
||||
CD86 |
4.14 |
2.21 |
1.44 |
108.3 |
||||
104.79 |
ISO |
1.97 |
- |
1.55 |
1.6 |
- |
109.4 |
|
CD54 |
3.26 |
1.29 |
1.61 |
115.2 |
||||
CD86 |
4.14 |
2.17 |
1.65 |
106.4 |
||||
125.75 |
ISO |
2.02 |
- |
1.70 |
1.5 |
- |
115.4 |
|
CD54 |
3.37 |
1.35 |
1.45 |
120.5 |
||||
CD86 |
4.22 |
2.20 |
1.41 |
107.8 |
||||
150.9 |
ISO |
2.04 |
- |
2.46 |
1.9 |
- |
90.1 |
|
CD54 |
3.33 |
1.29 |
2.01 |
115.2 |
||||
CD86 |
4.59 |
2.55 |
1.37 |
125.0 |
||||
181.08 |
ISO |
2.10 |
- |
2.91 |
2.6 |
- |
68.3 |
|
CD54 |
4.10 |
2.00 |
2.66 |
178.6 |
||||
CD86 |
6.45 |
4.35 |
2.13 |
213.2* |
||||
217.3 |
ISO |
2.08 |
- |
2.58 |
2.2 |
- |
78.2 |
|
CD54 |
3.88 |
1.80 |
2.20 |
160.7 |
||||
CD86 |
6.45 |
4.37 |
1.95 |
214.2* |
||||
260.76 |
ISO |
2.14 |
- |
2.93 |
2.5 |
- |
69.9 |
|
CD54 |
3.92 |
1.78 |
2.66 |
158.9 |
||||
CD86 |
7.33 |
5.19 |
1.94 |
254.4* |
*: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).
Table 4. Results of the second h-CLAT run
|
Concentration [µg/mL] |
Antibody / ISO |
MFI GeoMean (FITC) |
MFI - ISO |
Csto(Geo) GeoMean (7-AAD) |
Mean Cyto |
RFI (%) |
Viability (%) |
Negative control |
- |
ISO |
1.86 |
- |
1.87 |
1.9 |
- |
100 |
CD54 |
2.54 |
0.68 |
1.97 |
100 |
||||
CD86 |
3.84 |
1.98 |
1.72 |
100 |
||||
Vehicle control |
- |
ISO |
1.81 |
- |
1.84 |
1.7 |
- |
100 |
CD54 |
2.55 |
0.74 |
1.53 |
100 |
||||
CD86 |
3.93 |
2.12 |
1.77 |
100 |
||||
Positive control |
2 |
ISO |
2.14 |
- |
2.53 |
1.8 |
- |
93.8 |
CD54 |
5.74 |
3.6 |
1.57 |
486.5* |
||||
CD86 |
9.74 |
7.6 |
1.38 |
358.5* |
||||
3 |
ISO |
2.1 |
- |
1.97 |
1.6 |
- |
107.5 |
|
CD54 |
6.29 |
4.19 |
1.53 |
566.2* |
||||
CD86 |
9.2 |
7.1 |
1.28 |
334.9* |
||||
Test substance |
72.77 |
ISO |
1.97 |
- |
2.82 |
2.2 |
- |
76.8 |
CD54 |
2.66 |
0.69 |
2.46 |
93.2 |
||||
CD86 |
3.58 |
1.61 |
1.41 |
75.9 |
||||
87.33 |
ISO |
1.9 |
- |
1.99 |
1.7 |
- |
102.4 |
|
CD54 |
2.5 |
0.6 |
1.67 |
81.1 |
||||
CD86 |
3.65 |
1.75 |
1.36 |
82.5 |
||||
104.79 |
ISO |
1.93 |
- |
1.61 |
1.6 |
- |
108.9 |
|
CD54 |
2.6 |
0.67 |
1.58 |
90.5 |
||||
CD86 |
3.5 |
1.57 |
1.53 |
74.1 |
||||
125.75 |
ISO |
2.03 |
- |
1.55 |
1.5 |
- |
115.2 |
|
CD54 |
2.88 |
0.85 |
1.5 |
114.9 |
||||
CD86 |
3.97 |
1.94 |
1.41 |
91.5 |
||||
150.9 |
ISO |
2.05 |
- |
1.72 |
1.5 |
- |
111.3 |
|
CD54 |
2.98 |
0.93 |
1.65 |
125.7 |
||||
CD86 |
4.58 |
2.53 |
1.25 |
119.3 |
||||
181.08 |
ISO |
2.02 |
- |
1.78 |
1.7 |
- |
102.6 |
|
CD54 |
2.89 |
0.87 |
1.8 |
117.6 |
||||
CD86 |
4.14 |
2.12 |
1.43 |
100 |
||||
217.3 |
ISO |
2.02 |
- |
2.47 |
1.9 |
- |
91.8 |
|
CD54 |
2.87 |
0.85 |
1.74 |
114.9 |
||||
CD86 |
3.9 |
1.88 |
1.39 |
88.7 |
||||
260.76 |
ISO |
2.36 |
- |
2.87 |
2.4 |
- |
70.7 |
|
CD54 |
3.44 |
1.08 |
2.56 |
145.9 |
||||
CD86 |
7.16 |
4.8 |
1.84 |
226.4* |
*: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).
In the first run the relative fluorescence intensity of CD86 exceeded the threshold of 150% in the cells treated with test substance concentration between 181.08 and 260.76 μg/mL. In the second run the relative fluorescence intensity of CD86 exceeded the threshold at 260.76 μg/mL test substance concentration. Even if a higher test substance concentration was used in the main experiment as recommended according to the guideline, the result is considered to be valid and acceptable since the cell viability at this test substance concentration did not fall below 50% cell viability. In both independently performed experiments, the RFI value for CD54 did not exceed the threshold level of 200%.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
The skin sensitising potential of the test substance was determined by a Direct Peptide Reactivity Assay according to OECD 442C and in compliance with GLP (2016). In this study the test substance showed no or minimal peptide reactivity.
In a second study, the skin sensitizing potential of the test substance was determined by an ARE-Nrf2 Luciferase Test according to OECD 442D and in compliance with GLP (2016). In this study the test substance did not induce luciferase activity in the transgenic KeratinoSens™ cell line.
In a third study, the skin sensitising potential of the test substance was determined by a Human Cell Line Activation Test according to OECD 442E and in compliance with GLP (2016). Based on the obtained results, the test substance increasd the expression of CD86, a cell surface marker associated with the process of activation of monocytes and dendritic cells. Thus, the h-CLAT prediction is considered positive.
Based on a weight of evidence approach considering one positive and two negative results in the in vitro test battery according to the Adverse Outcome Pathway defined for skin sensitisation, 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 sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are conclusive but not sufficient for classification.
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