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Diss Factsheets

Toxicological information

Skin sensitisation

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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:
2018
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

Constituent 1
Chemical structure
Reference substance name:
Adenine
EC Number:
200-796-1
EC Name:
Adenine
Cas Number:
73-24-5
Molecular formula:
C5H5N5
IUPAC Name:
adenine

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.