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EC number: 288-098-3 | CAS number: 85650-88-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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Endpoint summary
Administrative data
Description of key information
Based on a weight of evidence evaluation of the available data from three in vitro and in chemico assays covering two of three key events, the test item is considered to not be a skin sensitiser:
-
DPRA: negative
-
Keratinosens: negative
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:
- 2020-01-28
- 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))
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- Skin sensitisation (In chemico test system)
Formulation of the Test Item
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.1479 g ± 10 %) based on the molecular weight and purity of the substance with the equation below. 0.1479 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.1478 g test chemical was weighted for the stock solution used for lysine peptide depletion determination in the runs.
HPLC conditions:
HPLC: Shimadzu LC-2030i Prominence
Serial number: L21445402951AE
Detector: 220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 μm)
Serial number: USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7μL
System equilibration: running mobile phase A and mobile phase B in a ratio of 1:1 for 2 hours at 30°C column temperature and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow
Mobile phases for HPLC:
Mobile Phase A – 0.100 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile
Positive control: CINNAMALDEHYDE - Key result
- Run / experiment:
- other: mean out of three replicates
- Parameter:
- other: mean peptide depletion cysteine
- Value:
- 9.68
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: mean out of three replicates
- Parameter:
- other: mean peptide depletion lysine
- Value:
- 0.02
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.
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:
- GHS criteria not met
- Conclusions:
- Based on these results a direct peptide reactivity assay (DPRA) according to OECD 442C the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitizer under the experimental conditions of the study.
- Executive summary:
In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA).
For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion resulted from the positive control cinnamaldehyde was 73.67 % ± 0.94 % with cysteine peptide and 62.24 % ± 3.78 % with the lysine peptide.
The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.0 % and 0.2 % percentages for the cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.68 % ± 2.26 % while the percent lysine peptide depletion was 0.02 % ± 0.44 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 4.85 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.
Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides. It is thus not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2019-12-04
- 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)
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Details on the study design:
- Skin sensitisation (in vitro test system)
Procedure of the KeratinoSens™ method
0. Preincubation of cells
1. Seeding of cells for testing - 24 h incubation
2. Solubility testing Preparation of the stock solution
3. Preparation of master plates
4. Exposure – 48 h incubation
5. Luciferase activation measurement
6. Cytotoxicity assessment
Formulation of the Test Item
Based on the test item stock solutions made of sterile ultrapure water, that was sterilized by filtering via 0.22 μm membrane filter, 2-fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test item creating a 100 × master plate. The 100 × master concentrations were further diluted 25-fold into exposure medium to obtain the 4 × master plate, by adding 10 μL of the 100 × master concentrations to 230 μL exposure medium and correcting DMSO concentration by adding 10 μL DMSO to all twelve 4 × master solutions.
Preparation of cells
Cells were subcultured upon reaching 80 - 90 % confluence and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.
Exposure
After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 μL of 4× master solution to 150 μL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.
Luciferase activity measurements
After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 μL), and 1× lysis buffer (20 μL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 μL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds. - Key result
- Run / experiment:
- other: based on two runs
- Parameter:
- other: Statistically significant induction over 1.5-fold
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: based on two runs
- Parameter:
- other: Viability ≥ 70 % at lowest concentration with ≥ 1.5-fold
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: based on two runs
- Parameter:
- other: EC1.5 (μM)
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in the OECD Test Guideline 442D, our laboratory demonstrated technical proficiency (Study Number: 392-442-4012), using the 10 Proficiency Substances listed in APPENDIX IA - ANNEX 1 of TG 442D. Moreover, a historical database of data generated with the positive control is maintained over time to confirm the reproducibility of the test method in the laboratory.
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:
- GHS criteria not met
- Conclusions:
- Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
- Executive summary:
In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
For the test item and positive control substance, in order to derive a prediction two independent tests were conducted. Since the results of the two runs were concordant, a third run was not needed to derive a conclusion.
The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.
For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in the tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests were the IC30 and IC50 values calculated.
Both tests were concluded negative, meaning that induction values for the test item did not exceed the 1.5-fold threshold. Therefore EC1.5 values were not determined. Moreover, no dose response could be observed in any of the tests.
Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
Referenceopen allclose all
Table 1 Mean peptide depletion values for the positive control and the test item
Name, replicate number |
Obtained mean % cysteine |
Obtained mean |
Mean % |
test item |
9.68 |
0.02 |
4.85 |
CINNAMALDEHYDE |
73.67 |
62.24 |
67.96 |
The average percent peptide depletion was calculated for the test item. Since no co-elution was observed, applying the cysteine 1:10 / lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. The mean percent peptide depletion value was 4.85 %. Thus, the test item is considered to be negative in the DPRA and classified in the no or minimal reactivity class when using the cysteine 1:10 / lysine 1:50 prediction model.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
There are two of three in vitro/in chemico studies available with the test item assessing its skin sensitising potential. The studies were conducted according to GLP and the respective OECD TG (OECD 442C, OECD 442D) and are both considered valid. The available data is considered suitable and reliable to allow assessment of the test items properties in regards to skin sensitisation.
OECD 442 C (DPRA)
In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA).
For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion resulted from the positive control cinnamaldehyde was 73.67 % ± 0.94 % with cysteine peptide and 62.24 % ± 3.78 % with the lysine peptide.
The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM – 0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.0 % and 0.2 % percentages for the cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.68 % ± 2.26 % while the percent lysine peptide depletion was 0.02 % ± 0.44 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 4.85 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.
Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides. It is thus not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.
OECD 442 D (KeratinoSense)
In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
For the test item and positive control substance, in order to derive a prediction two independent tests were conducted. Since the results of the two runs were concordant, a third run was not needed to derive a conclusion.
The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.
For the test item, twelve doses ranging from 2000 μM to 0.98 μM were used in the tests. The test item induced no cytotoxicity (viability < 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Thus, in none of the tests were the IC30 and IC50 values calculated.
Both tests were concluded negative, meaning that induction values for the test item did not exceed the 1.5-fold threshold. Therefore EC1.5 values were not determined. Moreover, no dose response could be observed in any of the tests.
Based on these results and the KeratinoSens™ prediction model, the test item was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
Conclusion
In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone validation using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).
Based on the results of this validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from theDPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90 % and an accuracy of 90 %.
In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al. 2012; Table 3). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.
Table 3: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.
DPRA
LuSens/
KeratinoSensTM
MUSST/
h-CLAT
Test battery evaluation
positive
positive
positive
sensitizer
positive
positive
negative
sensitizer
positive
negative
positive
sensitizer
positive
negative
negative
non-sensitizer
negative
positive
positive
sensitizer
negative
positive
negative
non-sensitizer
negative
negative
positive
non-sensitizer
negative
negative
negative
non-sensitizer
In accordance with the published evaluation scheme (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is considered to be a skin sensitizer based on the following results:
- DPRA: negative
- Keratinosens: negative
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Classification,
Labelling, and Packaging Regulation (EC) No 1272/2008
The
available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Based on
available data on skin sensitising properties, the test item does not
require classification as skin sensitiser according to Regulation (EC)
No 1272/2008 (CLP).
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.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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