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EC number: 946-002-3 | CAS number: 1818916-02-7
- 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
- OECD 442C: negative
- OECD 442D: positive
- OECD 442E: negative but inconclusive due to logP > 3.5
Skin sensitisation was studied using GLP in vitro studies performed according to OECD TG 442C-E. The following results have been obtained:
The conflicting in vitro data are insufficient to conclude on the endpoint of skin sensitisation. Therefore, an additional in vivo study was performed.
The GLP comliant study according to OECD TG 429 showed no evidence of SI values increasing over 3 thus no potential for skin sensitisation.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Dec 18, 2019 - Apr 02, 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
- Species:
- mouse
- Strain:
- CBA:J
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Inbred, SPF-Quality
- Age at study initiation: 10 weeks
- Weight at study initiation: Pre-test and Main test: 19.9 to 25.9 g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Humidity (%): 42 - 50 %
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: day 1 To: day 6 - Vehicle:
- methyl ethyl ketone
- Concentration:
- 5, 10, and 35% (w/w)
- No. of animals per dose:
- 5
- Details on study design:
- RANGE FINDING TESTS:
Two test item concentrations were tested; a 25% and 35% concentration.
- Compound solubility: 35 % in MEK
- Irritation:yes (At a 35% test item concentration No signs of systemic toxicity were noted and only very slight irritation of the ears was observed in animals treated with 35% test item concentration.)
- Lymph node proliferation response: -
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: Stimulation index > 3 - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- Standard statistical methods have been applied for data processing.
- Positive control results:
- Conc. SI
0%: 1.0
5% 1.3
10% 1.4
25% 5.2 - Key result
- Parameter:
- SI
- Value:
- 1.5
- Test group / Remarks:
- Test Group: 5% in MEK
- Key result
- Parameter:
- SI
- Value:
- 1.3
- Test group / Remarks:
- Test Group: 10% in MEK
- Key result
- Parameter:
- SI
- Value:
- 0.4
- Test group / Remarks:
- Test Group: 35% in MEK
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
- Executive summary:
Objective
The objective of this study was to evaluate whether the test material induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.
Study Design
The study was carried out based on the guidelines described in:
• OECD, Section 4, Health Effects, No.429 (2010).
• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".
• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.
Test item concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 35% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Methylethylketone). Three days after the last exposure, all animals were injected with 3Hmethyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.
Results
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 35% were 1084, 974 and 321 DPM, respectively. The mean DPM/animal value for the vehicle control group was 744 DPM. The SI values calculated for the test item concentrations 5, 10 and 35% were 1.5, 1.3 and 0.4, respectively. Since there was no indication that the test item elicits a SI = 3 when tested up to 35%, the test material was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 35%.
Conclusion
Based on these results, the test material would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-01-07 to 2019-02-14
- 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))
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- direct peptide reactivity assay (DPRA)
- 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.
- Positive control results:
- The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.95%.
- Key result
- Run / experiment:
- other: cysteine run
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 1.13
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: lysine run
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0.15
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of 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: Data should be considered in the context of integrated approached such as IATA.
- Conclusions:
- In this study under the given conditions the test item showed minimal reactivity towards both peptides. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP. - Executive summary:
In the present study the test material was dissolved in DMF, based on the results of the pre-experiments, but DMF did not pass the validity criteria for the Reference Control C and was therefore excluded from the evaluation.
The solubility experiment was carried on and ethanol was found as solvent, too. Based on a molecular weight of 336 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item, including the co-elution control. Samples of the test item (including the co-elution control) were centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item turbidity and precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item, including the co-elution control. Samples of the test item (including the co-elution control) were centrifuged prior to the HPLC analysis. Phase separation (small droplets) was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis
Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.
The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.64%). Since a precipitation was observed, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, no firm conclusion on the lack of reactivity should be drawn from a negative result, therefore, no prediction can be made.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-12-19 to 2019-02-25
- 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)
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
- 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:
- The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item 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 sensitisers and non-sensitisers.
- Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.85 (experiment 1); 4.59 (experiment 2)).
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Value:
- 2.1
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 3.91 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 95.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity
- Value:
- 2.43
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 3.91 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 130.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Other effects / acceptance of results:
- Acceptance Criteria
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.
The controls fullfilled the validity criteria of the test. - Interpretation of results:
- other: Data should be considered in the context of an integrated approach such as IATA.
- Conclusions:
- In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item may be considered as sensitiser.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP. - Executive summary:
In the present study the test material was dissolved in THF. Based on a molecular weight of 336 g/mol a stock solution of 200 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment, a max luciferase activity (Imax) induction of 2.46 was determined at a test item concentration of 500 µM but the corresponding cell viability was 33.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.59) was found to be 0.98 µM, the corresponding cell viability was >70% (125.8%). The luciferase activity induction rises up to 2.10 at 3.91 µM, from then on the substance is cytotoxic. No EC1.5 value could be calculated.
In the second experiment, a max luciferase activity (Imax) induction of 2.6 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 82.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.85) was found to be 0.98 µM, the corresponding cell viability was >70% (119%). The luciferase activity induction rises up to 2.51 at 7.81 µM, from then on the substance is cytotoxic. The last concentration is viable again but that seems to be an artefact. No EC1.5 value could be calculated.
A 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 sensitiser.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-05-29 to 2019-07-04
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
- Qualifier:
- according to guideline
- Guideline:
- other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- activation of dendritic cells
- Details on the study design:
- The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
- Positive control results:
- The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (334% experiment 1; 312% experiment 2) and 200% for CD54 (374% experiment 1; 1360% experiment 2) were clearly exceeded.
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 98
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 416.67 µg/mL
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 93
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 289.35 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 134
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 416.67 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 88
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 347.22 µg/mL
- Other effects / acceptance of results:
- Acceptance criteria:
The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.
The test mets the acceptance criteria. - Interpretation of results:
- other: Data should be considered in the context of an integrated approach such as IATA.
- Conclusions:
- In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Since the Log KOW is >3.5 the result has to be considered as inconclusive.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. - Executive summary:
In the present study the test material was dissolved in THF at 250 mg/mL (maximum solubility). For the dose finding assay stock solutions with concentrations ranging from 250 mg/mL to 1.96 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.
Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:
500.00, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45, 139.54 µg/mL
In experiment 1 precipitation of the test item was observed for concentration steps 500.00 µg/mL to 289.35 µg/mL and in experiment 2 precipitation of the test item was observed for concentration step 500.00 µg/mL to 200.94 µg/mL when mixing the test item stock solutions with cell culture medium.
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 92.6% (CD86), 93.7% (CD54) and 97.0% (isotype IgG1 control) in the first experiment and to 96.2% (CD86), 92.5% (CD54) and 96.4% (isotype IgG1 control) in the second experiment.
The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item would be considered as non-sensitiser, however, since the Log KOWis >3.5 (Log KOW> 6.7) the result has to be considered as inconclusive.
Referenceopen allclose all
Calculation of Stimulation Indices per Dose Group
Test item concentration |
Group Calculation |
||
Mean DPM per animal (2 lymph nodes) |
SD |
S.I. |
|
MEK (Vehicle Control) |
744 |
145 |
1.0 |
5 % Test Item in MEK |
1084 |
158 |
1.5 |
10 % Test Item in MEK | 974 |
219 |
1.3 |
35 % Test Item in MEK | 321 |
103 |
0.4 |
Cysteine and Lysine Values of the Calibration Curve
Sample |
Cysteine Peptide |
Lysine Peptide |
||
Peak Area at 220 nm |
Peptide Concentration [mM] |
PeakArea at 220 nm |
Peptide Concentration [mM] |
|
STD1 |
17.4940 |
0.5340 |
15.0310 |
0.5340 |
STD2 |
8.7090 |
0.2670 |
7.5160 |
0.2670 |
STD3 |
4.3730 |
0.1335 |
3.7790 |
0.1335 |
STD4 |
2.1750 |
0.0667 |
1.8940 |
0.0667 |
STD5 |
1.0820 |
0.0334 |
0.9520 |
0.0334 |
STD6 |
0.5670 |
0.0167 |
0.4950 |
0.0167 |
STD7 |
0.0000 |
0.0000 |
0.0000 |
0.0000 |
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 |
4.8780 |
0.1491 |
70.09 |
69.94 |
0.43 |
0.62 |
4.8480 |
0.1482 |
70.27 |
||||
4.9820 |
0.1523 |
69.45 |
||||
Test Item |
16.2480 |
0.4964 |
0.53 |
1.13 |
0.52 |
45.89 |
16.1020 |
0.4919 |
1.42 |
||||
16.1010 |
0.4919 |
1.43 |
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 |
5.5200 |
0.1958 |
61.01 |
59.97 |
2.04 |
3.41 |
5.4810 |
0.1944 |
61.28 |
||||
6.0000 |
0.2128 |
57.61 |
||||
Test Item |
14.2380 |
0.5058 |
0.00 |
0.15 |
0.26 |
173.21 |
14.1410 |
0.5024 |
0.45 |
||||
14.2880 |
0.5076 |
0.00 |
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.
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 |
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 |
0.64 |
Minimal Reactivity |
(negative) |
1.13 |
Minimal Reactivity |
(negative) |
Positive Control |
64.95 |
High Reactivity |
positive |
69.94 |
Moderate Reactivity |
positive |
Results of the Cytotoxicity Measurement
Concentration [µM] |
Cell Viability [%] |
||||
Experiment 1 |
Experiment 2 |
Mean |
SD |
||
Solvent Control |
- |
100 |
100 |
100 |
0.0 |
Positive Control |
4.00 |
79.5 |
94.8 |
87.1 |
10.8 |
8.00 |
77.7 |
103.7 |
90.7 |
18.4 |
|
16.00 |
67.6 |
101.1 |
84.4 |
23.7 |
|
32.00 |
50.1 |
88.2 |
69.2 |
27.0 |
|
64.00 |
48.9 |
107.5 |
78.2 |
41.4 |
|
Test Item |
0.98 |
125.8 |
119.0 |
122.4 |
4.8 |
1.95 |
109.9 |
95.3 |
102.6 |
10.4 |
|
3.91 |
95.9 |
130.8 |
113.3 |
24.6 |
|
7.81 |
54.2 |
74.9 |
64.6 |
14.6 |
|
15.63 |
40.2 |
49.5 |
44.8 |
6.6 |
|
31.25 |
25.3 |
40.5 |
32.9 |
10.8 |
|
62.50 |
21.9 |
36.3 |
29.1 |
10.1 |
|
125.00 |
23.8 |
36.3 |
30.1 |
8.9 |
|
250.00 |
25.9 |
37.9 |
31.9 |
8.5 |
|
500.00 |
33.1 |
46.0 |
39.6 |
9.1 |
|
1000.00 |
27.2 |
53.6 |
40.4 |
18.7 |
|
2000.00 |
30.3 |
82.3 |
56.3 |
36.8 |
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 |
1.09 |
0.95 |
1.26 |
1.10 |
0.16 |
|
8.00 |
1.23 |
1.05 |
1.22 |
1.17 |
0.10 |
|
|
16.00 |
1.29 |
1.24 |
1.39 |
1.31 |
0.08 |
|
|
32.00 |
1.89 |
1.80 |
1.78 |
1.82 |
0.06 |
* |
|
64.00 |
4.43 |
3.75 |
3.37 |
3.85 |
0.54 |
* |
|
Test Item |
0.98 |
1.40 |
1.82 |
1.55 |
1.59 |
0.21 |
* |
1.95 |
1.68 |
1.98 |
1.77 |
1.81 |
0.15 |
* |
|
3.91 |
1.98 |
2.30 |
2.02 |
2.10 |
0.18 |
* |
|
7.81 |
2.28 |
2.43 |
2.28 |
2.33 |
0.09 |
* |
|
15.63 |
2.18 |
2.28 |
2.06 |
2.17 |
0.11 |
* |
|
31.25 |
2.32 |
2.33 |
2.05 |
2.23 |
0.16 |
* |
|
62.50 |
2.01 |
2.13 |
1.99 |
2.04 |
0.07 |
* |
|
125.00 |
2.07 |
2.52 |
2.18 |
2.25 |
0.23 |
* |
|
250.00 |
2.24 |
2.01 |
2.11 |
2.12 |
0.12 |
* |
|
500.00 |
2.43 |
2.68 |
2.27 |
2.46 |
0.21 |
* |
|
1000.00 |
2.03 |
2.08 |
1.96 |
2.03 |
0.06 |
* |
|
2000.00 |
2.03 |
2.25 |
1.88 |
2.05 |
0.18 |
* |
* = significant induction according to Student’s t-test, p<0.05
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 |
1.14 |
1.21 |
1.16 |
1.17 |
0.04 |
|
8.00 |
1.21 |
1.22 |
1.31 |
1.25 |
0.05 |
|
|
16.00 |
1.41 |
1.58 |
1.71 |
1.57 |
0.15 |
* |
|
32.00 |
2.29 |
2.21 |
2.17 |
2.23 |
0.06 |
* |
|
64.00 |
4.24 |
4.60 |
4.91 |
4.59 |
0.34 |
* |
|
Test Item |
0.98 |
2.01 |
1.71 |
1.81 |
1.85 |
0.15 |
* |
1.95 |
2.14 |
1.89 |
2.10 |
2.04 |
0.13 |
* |
|
3.91 |
2.41 |
2.40 |
2.47 |
2.43 |
0.04 |
* |
|
7.81 |
2.48 |
2.52 |
2.53 |
2.51 |
0.02 |
* |
|
15.63 |
2.53 |
2.45 |
2.44 |
2.47 |
0.05 |
* |
|
31.25 |
2.27 |
2.52 |
2.32 |
2.37 |
0.13 |
* |
|
62.50 |
2.40 |
2.44 |
2.38 |
2.41 |
0.03 |
* |
|
125.00 |
2.28 |
2.43 |
2.39 |
2.37 |
0.08 |
* |
|
250.00 |
2.24 |
2.51 |
2.37 |
2.37 |
0.14 |
* |
|
500.00 |
2.51 |
2.60 |
2.28 |
2.46 |
0.16 |
* |
|
1000.00 |
2.50 |
2.67 |
2.43 |
2.54 |
0.12 |
* |
|
2000.00 |
2.57 |
2.79 |
2.46 |
2.60 |
0.17 |
* |
* = significant induction according to Student’s t-test, p<0.05
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 |
1.10 |
1.17 |
1.13 |
0.05 |
|
8.00 |
1.17 |
1.25 |
1.21 |
0.06 |
|
|
16.00 |
1.31 |
1.57 |
1.44 |
0.18 |
|
|
32.00 |
1.82 |
2.23 |
2.02 |
0.29 |
* |
|
64.00 |
3.85 |
4.59 |
4.22 |
0.52 |
* |
|
Test Item |
0.98 |
1.59 |
1.85 |
1.72 |
0.18 |
* |
1.95 |
1.81 |
2.04 |
1.93 |
0.16 |
* |
|
3.91 |
2.10 |
2.43 |
2.26 |
0.23 |
* |
|
7.81 |
2.33 |
2.51 |
2.42 |
0.13 |
* |
|
15.63 |
2.17 |
2.47 |
2.32 |
0.21 |
* |
|
31.25 |
2.23 |
2.37 |
2.30 |
0.10 |
* |
|
62.50 |
2.04 |
2.41 |
2.23 |
0.26 |
* |
|
125.00 |
2.25 |
2.37 |
2.31 |
0.08 |
* |
|
250.00 |
2.12 |
2.37 |
2.25 |
0.18 |
* |
|
500.00 |
2.46 |
2.46 |
2.46 |
0.00 |
* |
|
1000.00 |
2.03 |
2.54 |
2.28 |
0.36 |
* |
|
2000.00 |
2.05 |
2.60 |
2.33 |
0.39 |
* |
* = significant induction according to Student’s t-test, p<0.05
Additional Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Mean |
SD |
EC1.5[µM] |
n.a. |
n.a. |
n.a. |
n.a. |
Imax |
2.46 |
2.60 |
2.53 |
0.10 |
IC30[µM] |
6.33 |
15.48 |
10.91 |
6.47 |
IC50[µM] |
10.17 |
9.32 |
9.75 |
0.60 |
n.a.: not applicable
Acceptance Criteria
Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
CV Solvent Control PC (1% DMSO) |
< 20% |
11.3 |
pass |
8.2 |
pass |
CV Solvent Control TI (1% THF) |
<20% |
13.7 |
pass |
10.2 |
pass |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
2.0 |
pass |
3.0 |
pass |
EC1.5 PC |
± 2 x SD of historical mean |
21.99 |
pass |
14.31 |
pass |
Induction PC at 64 µM |
2 .00 < x < 8.00 |
3.85 |
pass |
4.59 |
pass |
Historical Data
Acceptance Criterion |
Range |
Mean |
SD |
N |
CV Solvent Control |
< 20% |
11.6 |
3.5 |
96 |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
2.4 |
0.6 |
96 |
EC1.5 PC |
7 < x < 34 µM |
18.5 |
6.0 |
96 |
Induction PC at 64 µM |
2.00 < x < 8.00 |
3.8 |
1.5 |
96 |
Results of the Cell Batch 6 Activation Test
Sample |
Concentration |
CD86 |
CD54 |
Activated |
Pass /Fail |
||||
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
yes/no |
|||
DNCB |
4 µg/mL |
85.4 |
327 |
>150 |
85.6 |
287 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
86.7 |
251 |
>150 |
86.5 |
269 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
96.5 |
76 |
≤150 |
96.6 |
77 |
≤200 |
no |
pass |
Results of the Cell Batch 7 Activation Test
Sample |
Concentration |
CD86 |
CD54 |
Activated |
Pass /Fail |
||||
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
Cell Viability [%] |
RFI |
Threshold OECD TG 442E |
yes/no |
|||
DNCB |
4 µg/mL |
86.9 |
422 |
>150 |
85.4 |
647 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
89.1 |
254 |
>150 |
90.0 |
454 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
97.9 |
53 |
≤150 |
98.0 |
70 |
≤200 |
no |
pass |
Results of the Dose Finding Assay
Sample |
|
Experiment 1 |
|
|
Concentration applied [µg/mL] |
Cell Viability [%] |
|
Medium Control |
-- |
-- |
97.20 |
Solvent Control |
THF |
-- |
96.90 |
Test Material |
C8 |
3.91 |
96.00 |
C7 |
7.81 |
95.80 |
|
C6 |
15.63 |
96.00 |
|
C5 |
31.25 |
95.10 |
|
C4 |
62.50 |
96.20 |
|
C3 |
125.00 |
95.20 |
|
C2 |
250.00 |
94.50 |
|
C1 |
500.00 |
94.90 |
|
Calculated CV75 [µg/mL] |
No CV75 |
||
Mean CV75 [µg/mL] |
No CV75 |
||
SD CV 75 [µg/mL] |
No SD |
CD54 and CD86 Expression Experiment 1
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
96.3 |
96.5 |
96.7 |
2363 |
1253 |
855 |
1508 |
398 |
102 |
94 |
276 |
147 |
Solvent Control 2 (THF) |
0.20% |
96.6 |
96.4 |
96.4 |
2202 |
1353 |
866 |
1336 |
487 |
100 |
100 |
254 |
156 |
Solvent Control 1 (DMSO) |
0.20% |
96.1 |
96.7 |
96.8 |
2307 |
1248 |
825 |
1482 |
423 |
100 |
100 |
280 |
151 |
DNCB |
4.00 |
72.9 |
70.7 |
69.0 |
5846 |
2477 |
893 |
4953 |
1584 |
334 |
374 |
655 |
277 |
Test Material |
500 |
92.6 |
93.7 |
97.0 |
1797 |
1223 |
861 |
936 |
362 |
70 |
74 |
209 |
142 |
416.67 |
93.1 |
94.6 |
96.9 |
2148 |
1180 |
834 |
1314 |
346 |
98 |
71 |
258 |
141 |
|
347.22 |
92.8 |
94.3 |
94.1 |
1955 |
1176 |
810 |
1145 |
366 |
86 |
75 |
241 |
145 |
|
289.35 |
94.0 |
92.8 |
94.4 |
1938 |
1264 |
810 |
1128 |
454 |
84 |
93 |
239 |
156 |
|
241.13 |
93.1 |
92.6 |
92.3 |
1888 |
1177 |
822 |
1066 |
355 |
80 |
73 |
230 |
143 |
|
200.94 |
92.7 |
92.7 |
92.1 |
2127 |
1148 |
825 |
1302 |
323 |
97 |
66 |
258 |
139 |
|
167.45 |
94.9 |
92.2 |
92.2 |
1922 |
1152 |
778 |
1144 |
374 |
86 |
77 |
247 |
148 |
|
139.54 |
94.3 |
93.5 |
91.4 |
1952 |
1189 |
768 |
1184 |
421 |
89 |
86 |
254 |
155 |
CD54 and CD86 Expression Experiment 2
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
97.5 |
97.0 |
97.3 |
1632 |
1001 |
795 |
837 |
206 |
92 |
127 |
205 |
126 |
Solvent Control 2 (THF) |
0.20% |
97.0 |
96.9 |
97.1 |
1581 |
1051 |
874 |
707 |
177 |
100 |
100 |
181 |
120 |
Solvent Control 1 (DMSO) |
0.20% |
96.3 |
96.6 |
97.4 |
1779 |
1035 |
873 |
906 |
162 |
100 |
100 |
204 |
119 |
DNCB |
4.0 |
71.5 |
73.8 |
75.2 |
3760 |
3139 |
935 |
2825 |
2204 |
312 |
1360 |
402 |
336 |
Test Material |
500.00 |
96.2 |
92.5 |
96.4 |
1840 |
1026 |
1097 |
743 |
-71 |
105 |
-40 |
168 |
94 |
416.67 |
95.8 |
95.6 |
96.7 |
1942 |
1043 |
998 |
944 |
45 |
134 |
25 |
195 |
105 |
|
347.22 |
95.5 |
95.8 |
95.9 |
1578 |
1023 |
867 |
711 |
156 |
101 |
88 |
182 |
118 |
|
289.35 |
96.3 |
96.1 |
96.2 |
1417 |
980 |
859 |
558 |
121 |
79 |
68 |
165 |
114 |
|
241.13 |
96.4 |
96.6 |
96.0 |
1713 |
1060 |
926 |
787 |
134 |
111 |
76 |
185 |
114 |
|
200.94 |
96.3 |
96.0 |
96.2 |
1618 |
1096 |
968 |
650 |
128 |
92 |
72 |
167 |
113 |
|
167.45 |
95.5 |
96.6 |
96.1 |
1563 |
1019 |
951 |
612 |
68 |
87 |
38 |
164 |
107 |
|
139.54 |
95.9 |
96.3 |
96.8 |
1348 |
1029 |
1001 |
347 |
28 |
49 |
16 |
135 |
103 |
Acceptance Criteria
Acceptance Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
||||
cell viability solvent controls [%] |
>90 |
96.3 |
- |
96.7 |
pass |
96.9 |
- |
97.5 |
pass |
number of test dosed with viability >50% CD86 |
≥4 |
8 |
pass |
8 |
pass |
||||
number of test dosed with viability >50% CD54 |
≥4 |
8 |
pass |
8 |
pass |
||||
number of test dosed with viability >50% IgG1 |
≥4 |
8 |
pass |
8 |
pass |
||||
RFI of positive control of CD86 |
≥150 |
334 |
pass |
312 |
pass |
||||
RFI of positive control of CD54 |
≥200 |
374 |
pass |
1360 |
pass |
||||
RFI of solvent control of CD86 |
<150 |
98 |
pass |
108 |
pass |
||||
RFI of solvent control of CD54 |
<200 |
106 |
pass |
51 |
pass |
||||
MFI ratio CD86/IgG1 for medium control [%] |
>105 |
89 |
pass |
84 |
pass |
||||
MFI ratio CD86/IgG1 for DMSO control [%] |
>105 |
122 |
pass |
56 |
pass |
||||
MFI ratio CD54/IgG1for medium control [%] |
>105 |
276 |
pass |
205 |
pass |
||||
MFI ratio CD54/IgG1for DMSO control [%] |
>105 |
280 |
pass |
204 |
pass |
Historical Data
Criterion |
mean |
SD |
N |
cell viability solvent controls [%] |
96.2 |
1.6 |
216 |
number of test doses with viability >50% |
- |
- |
555 |
RFI of positive control of CD86 |
358.9 |
90.1 |
36 |
RFI of positive control of CD54 |
435.7 |
285.8 |
36 |
RFI of THF control of CD86 |
102.9 |
17.4 |
36 |
RFI of THF control of CD54 |
102.0 |
15.4 |
36 |
MFI ratio IgG1/CD86 for medium control [%] |
285.6 |
124.3 |
36 |
MFI ratio IgG1/CD86 for THF control [%] |
270.8 |
105.0 |
36 |
MFI ratio IgG1/CD86 for DMSO control [%] |
221.6 |
58.5 |
112 |
MFI ratio IgG1/CD54 for medium control [%] |
308.6 |
137.1 |
36 |
MFI ratio IgG1/CD54 for THF control [%] |
158.2 |
28.4 |
36 |
MFI ratio IgG1/CD54 for DMSO control [%] |
147.7 |
25.6 |
112 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for skin sensitisation according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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