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Diss Factsheets
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EC number: 949-344-1 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30.03.2020 - 29.05.2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Version / remarks:
- 2018
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™
- Version / remarks:
- 2018
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- activation of keratinocytes
Test material
- Reference substance name:
- L-alanyl-L-tyrosine dihydrate
- Cas Number:
- 2219303-86-1
- Molecular formula:
- C12H16N2O4.2 H2O
- IUPAC Name:
- L-alanyl-L-tyrosine dihydrate
- Test material form:
- solid
Constituent 1
In vitro test system
- Details on the study design:
- PREPARATION OF TEST ITEM SOLUTIONS
- The test item was suspended in DMSO at 50 mM (white homogenous suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 500, 250, 500, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 µM (final concentration DMSO of 1%). At concentrations of 1.6 mM and higher the test item formed a suspension in DMSO whereas at 0.78 mM and lower it was fully soluble.
- All concentrations of the test item were tested in triplicate.
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- Test item concentrations were used within 2.5 hours after preparation.
TEST SYSTEM
- A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland).
- All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 – 37.0°C).
- For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator.
- The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0°C in the presence of 5% CO2.
LUCIFERASE ACTIVITY MEASUREMENT
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
CYTOTOXICITY ASSESSMENT
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
Results and discussion
- Positive control results:
- Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.62 and the EC1.5 was 42 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.84 and the EC1.5 was 58 µM.
In vitro / in chemico
Resultsopen allclose all
- Run / experiment:
- other: 1
- Parameter:
- other: maximum luciferase activity induction (Imax)
- Value:
- 1.19
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: 2
- Parameter:
- other: maximum luciferase activity induction (Imax)
- Value:
- 1.11
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (42 µM and 58 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.62-fold and 2.48-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.6% and 5.9% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.
The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.19-fold and 1.11-fold in experiment 1 and 2 respectively. the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.
Any other information on results incl. tables
Overview EC1.5, Imax, IC30and IC50Values
|
EC1.5(µM) |
Imax |
IC30(µM) |
IC50(µM) |
Test item Experiment 1 |
NA |
1.19 |
NA |
NA |
Test item Experiment 2 |
NA |
1.11 |
NA |
NA |
Pos Control Experiment 1 |
42 |
3.62 |
NA |
NA |
Pos Control Experiment 2 |
58 |
2.48 |
NA |
NA |
NA = Not applicable
Applicant's summary and conclusion
- Interpretation of results:
- other: inconclusive
- Conclusions:
- L-Alanyl-L-Tyrosine (L-Ala-L-Tyr) is classified as inconclusive (based on the absence of a biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes at test concentrations < 1000 μM) under the experimental conditions described.
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