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EC number: 202-796-7 | CAS number: 99-87-6
- 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
Two key events of the Adverse Outcome Pathway (AOP) for skin sensitisation were investigated in a Direct Peptide Reactivity Assay (DPRA, OECD guideline 442C) and in an ARE-Nrf2 Luciferase Test (OECD guideline 442D) after treatment with the test item. The test item is considered not suitable for DPRA due to poor water solubility under the conditions of this study. Thus, this study was stopped after pre-experiments. In the ARE-Nrf2 Luciferase Test, the test substance did not activate the LuSens cells up to a concentration of 166 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation AOP. Using Derek Nexus v. 6.0.1, no skin sensitising properties of the test item were estimated. As the substance is within the applicability domain of the model, the estimation can be regarded as accurate. In summary, it can be concluded that the test substance has no sensitizing properties.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation, other
- Type of information:
- (Q)SAR
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- Please refer to the QMRF and QPRF files provided under the section attached justification.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- Estimates the skin sensitising properties of chemicals using structural alert relationships.
- GLP compliance:
- no
- Key result
- Parameter:
- other: alerts
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- QSAR predicted value. The substance is (not) within the applicability domain of the model.
- Interpretation of results:
- other: No prediction of skin sensitisation
- Conclusions:
- Using Derek Nexus v. 6.0.1, no skin sensitising properties of the test item were estimated. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.
- Executive summary:
The skin sensitising properties were estimated using Derek Nexus v. 6.0.1. No skin sensitising properties were estimated based on the described QSAR method (Derek, 2020).
The adequacy of a prediction depends on the following conditions:
a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;
b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;
c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;
d) the (Q)SAR model is relevant for the regulatory purpose.
For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.
Description of the prediction Model
The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file.
Assessment of estimation domain
The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2020-06-30 to 2020-08-27
- 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:
- 06-2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Details on the study design:
- CONTROLS
- Medium control: Treatment medium
- Solvent: DMSO (final concentration 1% (v/v) in Treatment Medium), purity: ≥ 99%
- Positive control: EGDMA (final concentration 120 μM), CAS 97-90-5, purity: ≥ 97.5%
- Negative control: Lactic acid (final concentration 5000 μM), CAS 50-21-5, purity: ~ 90%
TEST ITEM PREPARATION
- The test item was dissolved or stably dispersed/suspended in DMSO to prepare a stock solution with a concentration of 200 mM immediately before treatment
- Test concentrations:
Dose finding assay (MTT assay): 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM
Main experiments: 66.7, 80.1, 96.1, 115, 138 and 166 µM
CELL CULTURE
- Incubation conditions: at 37°C ± 1.5°C and 5 ± 0.5% CO2
- Passage numbers: Passage 11 in the cytotoxicity test and 13 in the LuSens test for the main experiments 1 and 2, respectively
- Culture medium:
Cultivation medium: DMEM culture medium with Penicillin/Streptomycin (1% v/v), FBS (10% v/v) and puromycin (0.005% v/v)
Seeding Medium: DMEM culture medium with FBS (10% v/v)
Treatment Medium: DMEM culture medium with FBS (1% v/v)
- Seeding: 100 μL cell suspension (9000-11000 cells/well) in seeding medium, incubation for 24 hours ± 30 minutes
EXPERIMENTAL PROCEDURE
Dose finding test (MTT assay):
- MTT solution: MTT stock solution (5mg/mL in DPBS) in Treatment medium (1:10 soultion)
- Treatment: 150 μL treatment medium was added per well and 50 μL of the test item dilutions, the solvent, negative and positive controls and the medium control were added to the wells, respectively. At the end of the incubation period of 48 ± 1 hours, the cell cultures were microscopically evaluated for morphological alterations, precipitation or phase separation.
- Measurement of cell viability: At the end of the incubation period, cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the MTT working solution were added to each treatment well and the cells were incubated for 3 hours ± 30 minutes. After rinsing the MTT working solution, the cells of each well were treated with 100 μL MTT lysis agent (Isopropanol with
0.04 N HCl) for at least 30 minutes, while gently shaking. Thereafter the microplate was transferred to a microplate reader equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).
Main experiments (LuSens and MTT assay):
- Replicates: 2
- Treatment: For each main experiment one 96 well microtiter plate was prepared for MTT assay and one for the luciferase activity measurement.150 μL of treatment medium was distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control were added into the corresponding wells. At the end of the incubation period of 48 ± 1 hours, the cell cultures
were microscopically evaluated for morphological alterations, precipitation or phase separation.
- Measurement of cell viability (MTT): As described for the dose finding test
- Measurement of the Luciferase activity: The Steady-Glo® Mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® Substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady-Glo®-Mix. At the end of the incubation period, the Treatment Medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+. Thereafter, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 seconds per well.
REASON FOR CHOICE OF SYSTEM
The LuSens cell-line is an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The plasmid contains a luciferase gene (reporter gene) which is under transcriptional control of an antioxidant response element (ARE) of the rat NQO1 gene. Genes dependent on the ARE such as NQO1 are known to be upregulated by contact sensitisers (OECD 442C). - Positive control results:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (Main experiment 1: 5.05; Main experiment 2: 4.69).
The positive control had a relative cell viability ≥ 70% as compared to the solvent control
(ME 1: 70.17%; ME 2: 90.97%). - Key result
- Run / experiment:
- other: Main experiment 2
- Parameter:
- other: x-fold luciferase induction
- Value:
- 0.42
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: Main experiment 1
- Parameter:
- other: x-fold luciferase induction
- Value:
- 1.32
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- The average luciferase activity induction were not statistically significant.
- Key result
- Run / experiment:
- other: Dose finding assay (cytotoxicity test)
- Parameter:
- other: CV75 value
- Value:
- 138.1
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: The CV75 value of the cytotoxicity test was calculated as 138.1 μM.
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes - Interpretation of results:
- other: no activation of keratinocytes
- Conclusions:
- In conclusion, the test item did not activate the LuSens cells up to a concentration of 166 μM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).
- Executive summary:
This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of the test item.
In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 uM up to the highest tested concentration of 2000 uM (threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 138.1 uM.
The test item was tested in 2 independent main experiments.
The following concentrations of the test item were tested in the main experiments:
66.7, 80.1, 96.1, 115, 138, 166 |uM.
After treatment with the test item for 48 ± 1 hours the luciferase induction was above (>) 1.5 fold compared to the solvent control in 2 consecutive non-cytotoxic tested concentrations in the main experiment 1. A T-Test was performed for the statistical evaluation of the two consecutive non-cytotoxic tested concentrations with a luciferase induction of > 1.5 fold. The average luciferase activity induction were not statistically significant.
In the main experiment 2 the luciferase induction was not above or equal to 1.5 fold compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations, which confirms the negative result of the main experiment 1. Therefore, the LuSens prediction is considered negative.
The acceptance criteria were met:
- The average luciferase activity induction obtained with the positive control, 120 uM EGDMA was > 2.5 (ME 1: 5.05; ME 2: 4.69).
- The positive control had a relative cell viability > 70% as compared to the solvent control (ME 1: 70.17%; ME 2: 90.97%).
- The average luciferase activity induction obtained with the negative control, 5000 uM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.14; ME 2: 0.70).
- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 5.4%; ME 2: 7.2%).
At least three test concentrations had a cell viability of at least 70% relative to the solvent controls. Moreover, since the result is considered negative, at least one concentration was cytotoxic, i.e. had a cell viability < 70%.
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2020-07
- 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:
- 2020-06-26
- Deviations:
- yes
- Remarks:
- pretest to OECD 442C (solubility)
- Principles of method if other than guideline:
- pretest to OECD 442C
- GLP compliance:
- no
- Remarks:
- pretest to OECD 442C (solubility)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- SOLUBILITY TEST
- Solvent: Acetonitrile
- Test item preparation: 100 mM of the test item was prepared in acetonitrile.
Test 1: 100 μL of the 100 mM solution were added to 1500 μl buffer pH 7.5 and 400 μL acetonitrile and incubated for 24 h at room temperature.
Test 2: 500 μL of the 100 mM solution were added to 1500 μl buffer pH 10.2 and incubated for 24 h at room temperature. - Key result
- Run / experiment:
- other: Test 2
- Parameter:
- other: turbidity
- Remarks on result:
- other: turbid, after 24 h at RT: turbid
- Key result
- Run / experiment:
- other: Test 1
- Parameter:
- other: turbidity
- Remarks on result:
- other: slightly turbid, after 24 h at RT: slightly turbid
- Interpretation of results:
- other: not applicable
- Conclusions:
- The test item is considered not suitable for the DPRA due to the poor water solubility of the test item under the conditions of this study.
- Executive summary:
This study was performed to assess the solubility of the test item to conduct a DPRA. An appropriate solvent should dissolve the test item completely. To test the solubility, a test item concentration of 100 mM was clearly resolved in acetonitrile. After addition to the buffers pH 7.5 and pH 10.2, turbid liquids were formed due to the poor water solubility of the test item. During a 24-hour incubation at room temperature, the test item settled to the bottom. After strong shaking, both formulations appeared turbid again.
Referenceopen allclose all
Table 1: Results of the dose finding assay (cytotoxicity test)
Treatment Group | Concentration | Absorbance (OD570) | Mean OD570 | SD OD570 | Mean OD570 blank corr. | Cell viability [%] | |||
Well 1 | Well 2 | Well 3 | |||||||
Blank |
| 0.021 |
|
|
|
|
|
| |
Solvent control |
|
|
|
| 0.473 | 0.03 | 0.452 | 100.0 | |
Medium control |
|
|
|
| 0.574 | 0.07 | 0.553 | 122.25 | |
Positive Control | 120 uM | 0.475 | 0.479 |
| 0.477 | 0.00 | 0.456 | 100.87 | |
Negative Control | 5000 uM | 0.414 | 0.440 | 0.427 | 0.427 | 0.01 | 0.406 | 89.81 | |
Test Item | C1 | 0.98 uM | 0.305 | 0.411 | 0.405 | 0.374 | 0.06 | 0.353 | 78.01 |
C2 | 1.95 uM | 0.329 | 0.349 | 0.411 | 0.363 | 0.04 | 0.342 | 75.65 | |
C3 | 3.91 uM | 0.403 | 0.426 | 0.441 | 0.423 | 0.02 | 0.402 | 89.00 | |
C4 | 7.81 uM | 0.417 | 0.421 | 0.436 | 0.425 | 0.01 | 0.404 | 89.29 | |
C5 | 15.6 uM | 0.432 | 0.439 | 0.429 | 0.433 | 0.01 | 0.412 | 91.21 | |
C6 | 31.3 uM | 0.401 | 0.439 | 0.434 | 0.425 | 0.02 | 0.404 | 89.29 | |
C7 | 62.5 uM | 0.353 | 0.388 | 0.409 | 0.383 | 0.03 | 0.362 | 80.15 | |
C8 | 125 uM | 0.359 | 0.413 | 0.408 | 0.393 | 0.03 | 0.372 | 82.36 | |
C9 | 250 uM | 0.018 | 0.065 | 0.142 | 0.075 | 0.06 | 0.054 | 11.94 | |
C10 | 500 uM | 0.017 | 0.017 | 0.018 | 0.017 | 0.00 | -0.004 | 0.00* | |
C11 | 1000 uM | 0.018 | 0.018 | 0.018 | 0.018 | 0.00 | -0.003 | 0.00* | |
C12 | 2000 uM | 0.018 | 0.018 | 0.019 | 0.018 | 0.00 | -0.003 | 0.00* |
The CV75 value of the cytotoxicity test was calculated as 138.1 µM
Table 2: Results of the main experiment 1 (cell viability)
Treatment Group | Concentration | Absorbance (OD570) | Mean OD570 | SD OD570 | Mean OD570 blank corr. | Cell viability [%] | ||||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | |||||||
Blank |
| 0.015 |
|
|
|
|
|
|
|
|
| |
Solvent control |
|
|
|
|
|
|
| 0.264 | 0.05 | 0.249 | 100.0 | |
Medium control |
|
|
|
|
|
|
| 0.278 | 0.03 | 0.263 | 105.40 | |
Positive Control | 120 uM | 0.157 | 0.213 | 0.176 | 0.201 | 0.202 |
| 0.190 | 0.02 | 0.175 | 70.17 | |
Negative Control | 5000 uM | 0.171 | 0.179 | 0.207 | 0.207 | 0.195 | 0.191 | 0.192 | 0.01 | 0.177 | 70.91 | |
Test Item | C1 | 66.7 uM | 0.248 | 0.228 | 0.237 |
|
|
| 0.238 | 0.01 | 0.223 | 89.38 |
C2 | 80.1 uM | 0.253 | 0.277 | 0.227 |
|
|
| 0.252 | 0.03 | 0.237 | 95.27 | |
C3 | 96.1 uM | 0.335 | 0.323 | 0.306 |
|
|
| 0.321 | 0.01 | 0.306 | 122.96 | |
C4 | 115.0 uM | 0.288 | 0.271 | 0.235 |
|
|
| 0.265 | 0.03 | 0.250 | 100.22 | |
C5 | 138.0 uM | 0.258 | 0.260 | 0.264 |
|
|
| 0.261 | 0.00 | 0.246 | 98.61 | |
C6 | 166.0 uM | 0.062 | 0.139 | 0.250 |
|
|
| 0.150 | 0.09 | 0.135 | 54.32 |
Table 3: Results of the main experiment 1 (fold induction)
Treatment Group | Concentration | Luminescence | Mean Luminescence | SD Luminescence | Mean Luminescence blank corr. | Fold Induction | ||||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | |||||||
Blank |
| 155 |
|
|
|
|
|
|
|
|
| |
Solvent control |
|
|
|
|
|
|
| 211.2 | 11.47 | 56.2 | 1.00 | |
Medium control |
|
|
|
|
|
|
| 217.4 | 17.16 | 62.4 | 1.11 | |
Positive Control | 120 uM | 473 | 451 | 436 | 414 | 421 |
| 439.0 | 23.76 | 284.0 | 5.05 | |
Negative Control | 5000 uM | 214 | 207 | 222 | 214 | 222 | 236 | 219.2 | 10.01 | 64.2 | 1.14 | |
Test Item | C1 | 66.7 uM | 399 | 229 | 214 |
|
|
| 280.7 | 102.75 | 125.7 | 2.24 |
C2 | 80.1 uM | 347 | 251 | 229 |
|
|
| 275.7 | 62.75 | 120.7 | 2.15 | |
C3 | 96.1 uM | 288 | 214 | 207 |
|
|
| 236.3 | 44.88 | 81.3 | 1.45 | |
C4 | 115.0 uM | 259 | 251 | 200 |
|
|
| 236.7 | 32.01 | 81.7 | 1.45 | |
C5 | 138.0 uM | 281 | 259 | 244 |
|
|
| 261.3 | 18.61 | 106.3 | 1.89 | |
C6 | 166.0 uM | 222 | 236 | 229 |
|
|
| 229.0 | 7.00 | 74.0 | 1.32 |
The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was 5.4% in the main experiment 1
Table 4: Results of the main experiment 2 (cell viability)
Treatment Group | Concentration | Absorbance (OD570) | Mean OD570 | SD OD570 | Mean OD570 blank corr. | Cell viability [%] | ||||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | |||||||
Blank |
| 0.021 |
|
|
|
|
|
|
|
|
| |
Solvent control |
|
|
|
|
|
|
| 0.452 | 0.06 | 0.431 | 100.0 | |
Medium control |
|
|
|
|
|
|
| 0.374 | 0.04 | 0.353 | 81.84 | |
Positive Control | 120 uM | 0.387 | 0.521 | 0.406 | 0.357 | 0.395 |
| 0.413 | 0.06 | 0.392 | 90.97 | |
Negative Control | 5000 uM | 0.365 | 0.384 | 0.419 | 0.444 | 0.339 | 0.414 | 0.394 | 0.04 | 0.373 | 86.56 | |
Test Item | C1 | 66.7 uM | 0.460 | 0.345 | 0.328 |
|
|
| 0.378 | 0.07 | 0.357 | 82.73 |
C2 | 80.1 uM | 0.382 | 0.386 | 0.353 |
|
|
| 0.374 | 0.02 | 0.353 | 81.80 | |
C3 | 96.1 uM | 0.439 | 0.326 | 0.437 |
|
|
| 0.401 | 0.06 | 0.380 | 88.06 | |
C4 | 115.0 uM | 0.483 | 0.459 | 0.354 |
|
|
| 0.432 | 0.07 | 0.411 | 95.33 | |
C5 | 138.0 uM | 0.021 | 0.017 | 0.017 |
|
|
| 0.018 | 0.00 | -0.003 | 0.00* | |
C6 | 166.0 uM | 0.017 | 0.017 | 0.017 |
|
|
| 0.017 | 0.00 | -0.004 | 0.00* |
Table 5: Results of the main experiment 2 (fold induction)
Treatment Group | Concentration | Luminescence | Mean Luminescence | SD Luminescence | Mean Luminescence blank corr. | Fold Induction | ||||||
Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | |||||||
Blank |
| 192 |
|
|
|
|
|
|
|
|
| |
Solvent control |
|
|
|
|
|
|
| 281.5 | 20.15 | 89.5 | 1.00 | |
Medium control |
|
|
|
|
|
|
| 288.5 | 18.74 | 96.5 | 1.08 | |
Positive Control | 120 uM | 576 | 613 | 606 | 658 | 606 |
| 611.8 | 29.52 | 419.8 | 4.69 | |
Negative Control | 5000 uM | 266 | 259 | 259 | 266 | 229 | 251 | 255.0 | 13.90 | 63.0 | 0.70 | |
Test Item | C1 | 66.7 uM | 443 | 288 | 266 |
|
|
| 332.3 | 96.47 | 140.3 | 1.57 |
C2 | 80.1 uM | 362 | 266 | 273 |
|
|
| 300.3 | 53.52 | 108.3 | 1.21 | |
C3 | 96.1 uM | 347 | 273 | 266 |
|
|
| 295.3 | 44.88 | 103.3 | 1.16 | |
C4 | 115.0 uM | 310 | 288 | 259 |
|
|
| 285.7 | 25.58 | 93.7 | 1.05 | |
C5 | 138.0 uM | 259 | 266 | 214 |
|
|
| 246.3 | 28.22 | 54.3 | 0.61 | |
C6 | 166.0 uM | 222 | 244 | 222 |
|
|
| 229.3 | 12.70 | 37.3 | 0.42 |
The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was 7.2% in the main experiment 2
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
DPRA, WoE
This study was performed to assess the solubility of the test item to conduct a DPRA. An appropriate solvent should dissolve the test item completely. To test the solubility, a test item concentration of 100 mM was clearly resolved in acetonitrile. After addition to the buffers pH 7.5 and pH 10.2, turbid liquids were formed due to the poor water solubility of the test item. During a 24-hour incubation at room temperature, the test item settled to the bottom. After strong shaking, both formulations appeared turbid again. Thus, this study was stopped after pre-experiments.
ARE-Nrf2 Luciferase Test, WoE
This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of the test item. In the cytotoxicity test, cytotoxic effects were observed following incubation with the test item starting with the concentration of 250 uM up to the highest tested concentration of 2000 uM (threshold of cytotoxicity: < 75%). The CV75 value of the cytotoxicity test was calculated as 138.1 uM. The test item was tested in 2 independent main experiments. The following concentrations of the test item were tested in the main experiments: 66.7, 80.1, 96.1, 115, 138, 166 |uM.
After treatment with the test item for 48 ± 1 hours the luciferase induction was above (>) 1.5 fold compared to the solvent control in 2 consecutive non-cytotoxic tested concentrations in the main experiment 1. A T-Test was performed for the statistical evaluation of the two consecutive non-cytotoxic tested concentrations with a luciferase induction of > 1.5 fold. The average luciferase activity induction were not statistically significant. In the main experiment 2 the luciferase induction was not above or equal to 1.5 fold compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations, which confirms the negative result of the main experiment 1. Therefore, the LuSens prediction is considered negative.
QSAR, WoE
The skin sensitising properties were estimated using Derek Nexus v. 6.0.1. No skin sensitising properties were estimated based on the described QSAR method (Derek, 2020).
The adequacy of a prediction depends on the following conditions:
- a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;
- b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;
- c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;
- d) the (Q)SAR model is relevant for the regulatory purpose.
For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.
Description of the prediction Model
The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file.
Assessment of estimation domain
The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.
Conclusion: Based on the ARE-Nrf2 Luciferase Test and the QSAR prediction, the test item is not considered to possess skin sensitising potential.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the available data, it is concluded that the test item does not have skin sensitising potential and therefore shall not be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.
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