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EC number: 200-796-1 | CAS number: 73-24-5
- 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
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- Endpoint summary
- Stability
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- 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:
- weight of evidence
- Study period:
- 2018-07-23 to 2018-09-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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:
- adopted June 25, 2018
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
- Version / remarks:
- 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- activation of keratinocytes
Test material
- Reference substance name:
- Adenine
- EC Number:
- 200-796-1
- EC Name:
- Adenine
- Cas Number:
- 73-24-5
- Molecular formula:
- C5H5N5
- IUPAC Name:
- adenine
Constituent 1
In vitro test system
- 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.
Cell line used: KeratinoSensTM (Givaudan, Switzerland)
Technical material and conditions:
- Maintenance Medium: D-MEM (GlutaMAXTM) with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM (GlutaMAXTM) with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS
- Test Item Exposure Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 1 % FBS
- Luciferace reagent: Luciferase Assay Substrate (Promega, Cat. No.: E1501)
- Assay Buffer: Luciferase Assay Buffer (Promega, Cat. No.: E1501)
- Lysisbuffer: Luciferase Cell Culture Lysis (Promega Cat. No.: E1531)
- MTT Solution: MTT stock solution: 5 mg/mL in DPBS
- SDS solution: 10% (w/v) SDS in dist. water
- DPBS solution: DPBS solution (without Ca2+/Mg2+)
Controls used:
- Vehicle control: DMSO: 1% (v/v) in test item exposure medium
- Positive control: Cinnamic aldehyde in DMSO, 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
- Blank control: vehicle control without cells
Test item: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 μM
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.
Test procedure:
Each concentration step of the test item (twelve concentrations, 0.49 - 1000 µM) and the positive control (5 concentrations, 4 - 64 µM) was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run. The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a plate reader. Cell viability was determined by a MTT assay. The test substance was incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 10% SDS solution the plate was incubated at 37 °C ± 1 °C and 5% CO2 overnight. The OD was measured at a wavelength of 600 nm.
Data analysis:
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consists of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run should be performed.
Acceptance criteria:
- 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.
Evaluation of results:
The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.
Results and discussion
- Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 5 and 7 (6.43 in experiment 1; 5.78 in experiment 2).
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity; Imax
- Value:
- 1.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 67.1
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Value:
- 761.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity, Imax
- Value:
- 1.82
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 69.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: concentration: 1000 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Value:
- 551.97
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
Any other information on results incl. tables
Table: Additional parameters
Parameter | Experiment 1 | Experiment 2 | Mean | SD |
EC1.5[MM] | 761.3 | 551.97 | 656.63 | 148.02 |
Imax | 1.7 | 1.82 | 1.76 | 0.09 |
IC30[MM] | 898 | 947.11 | 922.55 | 34.72 |
IC50[MM] | n/a | n/a | n/a | n/a |
n/a = not applicable
Table 2: Results of the Cytotoxicity Measurement
Concentration [MM] |
Cell Viability [%] | ||||
Experiment 1 | Experiment 2 | Mean | SD | ||
Solvent Control | - | 100 | 100 | 100 | 0.0 |
Positive Control | 4.00 | 86.4 | 103.6 | 95.0 | 12.2 |
8.00 | 92.4 | 105.9 | 99.2 | 9.5 | |
16.00 | 96 | 100 | 98.0 | 2.8 | |
32.00 | 86.6 | 101.3 | 93.9 | 10.4 | |
64.00 | 84.1 | 94.7 | 89.4 | 7.5 | |
Test Item | 0.49 | 96.4 | 77.6 | 87.0 | 13.3 |
0.98 | 105.5 | 79.5 | 92.5 | 18.4 | |
1.95 | 99.7 | 68.4 | 84.0 | 22.2 | |
3.91 | 86.1 | 75.8 | 80.9 | 7.3 | |
7.81 | 85.1 | 72.8 | 79.0 | 8.7 | |
15.63 | 85.9 | 73.1 | 79.5 | 9.0 | |
31.25 | 90.9 | 72.8 | 81.8 | 12.8 | |
62.50 | 84.6 | 71.2 | 77.9 | 9.5 | |
125.00 | 87.2 | 69.7 | 78.5 | 12.3 | |
250.00 | 83.2 | 73.1 | 78.1 | 7.1 | |
500.00 | 81.2 | 74.8 | 78.0 | 4.5 | |
1000.00 | 67.1 | 69.4 | 68.3 | 1.6 |
Table 3: Induction of Luciferase Activity Experiment 1
Experiment 1 | Concentration [MM] | 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 | 1.62 | 1.18 | 1.36 | 1.39 | 0.22 | |
8.00 | 1.27 | 1.20 | 1.14 | 1.20 | 0.07 | ||
16.00 | 1.49 | 1.63 | 1.53 | 1.55 | 0.08 | * | |
32.00 | 2.20 | 1.84 | 2.24 | 2.09 | 0.22 | * | |
64.00 | 7.00 | 6.59 | 5.69 | 6.43 | 0.67 | * | |
Test Item | 0.49 | 1.15 | 1.11 | 1.16 | 1.14 | 0.03 | |
0.98 | 1.09 | 1.07 | 1.18 | 1.11 | 0.06 | ||
1.95 | 1.07 | 1.03 | 0.91 | 1.00 | 0.08 | ||
3.91 | 1.10 | 1.10 | 1.20 | 1.13 | 0.06 | ||
7.81 | 1.16 | 1.17 | 1.39 | 1.24 | 0.13 | ||
15.63 | 1.19 | 1.14 | 1.22 | 1.18 | 0.04 | ||
31.25 | 1.26 | 1.32 | 1.25 | 1.28 | 0.04 | ||
62.50 | 1.31 | 1.26 | 1.20 | 1.26 | 0.05 | ||
125.00 | 1.28 | 1.16 | 1.28 | 1.24 | 0.07 | ||
250.00 | 1.41 | 1.46 | 1.20 | 1.36 | 0.14 | ||
500.00 | 1.30 | 1.20 | 1.36 | 1.29 | 0.08 | ||
1000.00 | 1.86 | 1.60 | 1.63 | 1.70 | 0.14 | * |
* = significant induction according to Student's t-test, p<0.05
Table 4: Induction of Luciferase Activity Experiment 2
Experiment 2 | Concentration [MM] | 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 | 1.34 | 1.28 | 1.24 | 1.29 | 0.05 | |
8.00 | 1.36 | 1.32 | 1.24 | 1.31 | 0.06 | ||
16.00 | 1.54 | 1.62 | 1.62 | 1.60 | 0.05 | * | |
32.00 | 2.02 | 2.16 | 2.21 | 2.13 | 0.10 | * | |
64.00 | 7.06 | 4.69 | 5.58 | 5.78 | 1.19 | * | |
Test Item | 0.49 | 1.14 | 1.50 | 1.14 | 1.26 | 0.21 | |
0.98 | 0.86 | 1.01 | 1.01 | 0.96 | 0.09 | ||
1.95 | 0.97 | 1.19 | 1.13 | 1.10 | 0.12 | ||
3.91 | 1.01 | 1.24 | 1.10 | 1.11 | 0.11 | ||
7.81 | 1.19 | 1.54 | 1.41 | 1.38 | 0.18 | ||
15.63 | 1.17 | 1.33 | 1.54 | 1.35 | 0.19 | ||
31.25 | 1.13 | 1.21 | 1.36 | 1.24 | 0.12 | ||
62.50 | 1.29 | 1.32 | 1.46 | 1.36 | 0.09 | ||
125.00 | 1.18 | 1.44 | 1.30 | 1.31 | 0.13 | ||
250.00 | 1.19 | 1.49 | 1.38 | 1.35 | 0.16 | ||
500.00 | 1.35 | 1.63 | 1.41 | 1.46 | 0.15 | ||
1000.00 | 1.82 | 1.73 | 1.92 | 1.82 | 0.10 | * |
* = significant induction according to Student's t-test, p<0.05
Table 5: Induction of Luciferase Activity - Overall Induction
Concentration [MM] | Fold Induction | Significance | ||||
Experiment 1 | Experiment 2 | Mean | SD | |||
Solvent Control | - | 1.00 | 1.00 | 1.00 | 0.00 | - |
Positive Control | 4.00 | 1.39 | 1.29 | 1.34 | 0.07 | |
8.00 | 1.20 | 1.31 | 1.26 | 0.07 | ||
16.00 | 1.55 | 1.60 | 1.57 | 0.03 | * | |
32.00 | 2.09 | 2.13 | 2.11 | 0.03 | * | |
64.00 | 6.43 | 5.78 | 6.10 | 0.46 | * | |
Test Item | 0.49 | 1.14 | 1.26 | 1.20 | 0.09 | |
0.98 | 1.11 | 0.96 | 1.04 | 0.11 | ||
1.95 | 1.00 | 1.10 | 1.05 | 0.06 | ||
3.91 | 1.13 | 1.11 | 1.12 | 0.01 | ||
7.81 | 1.24 | 1.38 | 1.31 | 0.10 | ||
15.63 | 1.18 | 1.35 | 1.27 | 0.11 | ||
31.25 | 1.28 | 1.24 | 1.26 | 0.03 | ||
62.50 | 1.26 | 1.36 | 1.31 | 0.07 | ||
125.00 | 1.24 | 1.31 | 1.27 | 0.05 | ||
250.00 | 1.36 | 1.35 | 1.36 | 0.00 | ||
500.00 | 1.29 | 1.46 | 1.37 | 0.13 | ||
1000.00 | 1.70 | 1.82 | 1.76 | 0.09 | * |
* = significant induction according to Student's t-test, p<0.05
Applicant's summary and conclusion
- Interpretation of results:
- other: no activation of keratinocytes
- Remarks:
- 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.
- Conclusions:
- In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline 442D, the test item did not induce luciferase activity.
- Executive summary:
In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD TG 442D, the skin sensitising potential of the test item was determined. The test item was dissolved in distilled water. Based on a molecular weight of 135.13 g/mol a stock solution of 100 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: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 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 1.70 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 67.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.70) was found to be 1000 µM. The corresponding cell viability was < 70% (67.1%). The calculated EC1.5 was <1000 µM (761.30). In the second experiment, a max luciferase activity (Imax) induction of 1.82 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 69.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.82) was found to be 1000 µM. The corresponding cell viability was < 70% (69.4%). The calculated EC1.5 was <1000 µM (551.97). Due to cell viability < 70% at the highest test item concentration and due to a missing dose response for luciferase activity induction in each individual run as well as for an overall luciferase activity induction the test item showed no sensitising potential. The controls confirmed the validity of the study.
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