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Diss Factsheets

Administrative data

Description of key information

The substance is not sensitizing to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-04-18 to 2017-04-28
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:
2015
Deviations:
yes
Remarks:
please refer to 'principles of method if other than guideline'
Qualifier:
according to guideline
Guideline:
other: OECD. (22. May 2015). PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS. ENV/JM/MONO(2015)6.
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: BASF SE: Protocol LuSens Assay, Last update: 16. May 2014
Version / remarks:
16. May 2014
Deviations:
no
Principles of method if other than guideline:
The used cell line as well as the negative and the positive control is different in comparison to the OECD 442D guideline. Also the test performance differs in details (e.g. performance of a CRFT, amount of tested concentrations of controls, incubation period of MTT solution or lysis buffer).
Prior to routine use, the validity of the LuSens test at the test facility was demonstrated in a proficiency study. For all control substances historical data are available, which demonstrates the reliability and the validity of those substances.
For this reason, all deviations of the LuSens test in comparison to the OECD 442D are declared as uncritical. The end result is not affected by those changes.
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
appropriate test method for determination of the skin sensitising potential
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 03/2016_T_R_JM
- Expiration date of the lot/batch: 26. Jun. 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5°C)
- Solubility of the test substance in the solvent/vehicle: The test item was soluble in DMSO at the required concentration of 200 mM.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution containing 200 mM (nominal) of the test item in DMSO was prepared and used for preparatrion of a geometric series of the subsequent concentrations. After slight shaking, the test item was completely solved. The stock solution as well as the dilutions were freshly prepared on the day of treatment.




Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:
The assay employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway. In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two, but a maximum of three independent and valid experiments will be performed. The assay is used for discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the subcategories 1 A and 1 B is not possible.
Positive control results:
All control substances indicated the expected effect. Regarding the Luciferase induction, the positive control induced a clear effect with an induction value of 6.8 fold in comparison to the solvent control.
Key result
Run / experiment:
other: II
Parameter:
other: induction of luciferase
Remarks:
fold induction compared to solvent control
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: II
Parameter:
other: cell viability [%]
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: I
Parameter:
other: induction of luciferase
Remarks:
fold induction compared to solvent control
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: I
Parameter:
other: cell viability [%]
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
Concerning the test item, no cytotoxic effect was observed at all test item concentrations when compared to the solvent control. The viability values were all ≥101%. Therefore all concentrations were analysable for luciferase induction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values > 99 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 0.9 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 6.8 fold in comparison to the solvent control. Concerning the test item, no cytotoxic effect was observed at all test item concentrations when compared to the solvent control. The viability values were all ≥ 101 %. Therefore all concentrations were analysable for luciferase induction. None of the tested concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Interpretation of results:
GHS criteria not met
Conclusions:
No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal concentration of the test item. Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor.
Executive summary:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined. In the experiments, the highest nominal applied concentration (2000 μM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in none of the experiments. DMSO (final concentration: 1 %) was used as solvent control. Lactic acid (5000 μM) was used as negative control and EGDMA (120 μM) as positive control. No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal concentration of the test item. Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
Instruments and Devices
Usual laboratory equipment and the following instruments were used in this study:
- Analytical and precison scales
- Volumetric measurement tools
- pH-meter
- Standard laboratory glassware
- Incubation chamber capable of holding 25 ± 2.5 °C

HPLC system:
Designation: HPLC_4
Components:
Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981
An ACE Excel SuperC18 150x3 mm column with 3 μm particles was used. This column was used instead the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline because it delivers substantially better peak shape for the peptides.

Peptides with ≥ 95 % purity, syntheseized by Genecust, Dudelange, Luxemburg, were used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 751.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 776.2 g/mol)

Chemicals
- Water for chromatography
- Demineralized water
- Acetonitrile for chromatography
- Trifluoroacetic acid
- Isopropanol
- Acetone
- Dimethylsulfoxide

Positive control
Positive controls are treated identically as the test item. The following positive controls were used:
- Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %) used as 100 mM solution in acetonitrile for the cysteine peptide, Depletion range 60.8 – 100 %
- 2,3-Butanedione (CAS 431-03-8, >97 %) will be used as 100 mM solution in acetonitrile for the lysine peptide, Depletion range 10 – 45 %
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing midrange depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution are prepared in triplicate (Sets A, B1, B2 and C, total 12 samples per peptide). Set A is analysed together with the peptide calibration standards, sets B1, B2 and C are incubated with the samples. Sets B1 and B2 are analysed at the start and end of the analysis sequence and are used as stability control for the peptide over the total analysis time. Set C is analysed together with the samples and is used for calculation of the peptide depletion.

Acceptance criteria
- The standard calibration curve should have an r² > 0.99
- The measured values of solvent control samples of sets A and C should be 0.5 ± 0.05 mM
- The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
- The mean peptide depletion value for the positive control cinnamic aldehyde should be 60.8 % - 100 % for the Cys-Peptide and 40.2 % - 69.0 % for the lysine peptide with a maximum standard deviation (SD) of < 14.9 % for the Cys-Peptide and < 11.6 % for the Lys-Peptide. If one of the acceptance criteria is not fulfilled, the test is repeated. If the result is unambiguous even though one of the criteria is not met, the test may be considered valid by the study director, but justification must be given.
If the mean percent depletion falls in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.
Key result
Run / experiment:
other: mean of three runs
Parameter:
other: mean peptide depletion of both peptides (%)
Value:
1.15
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: mean of three runs
Parameter:
other: Cys-Peptide depletion [%]
Value:
1.69
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: mean of three runs
Parameter:
other: Lys-Ppetide depletion [%]
Value:
0.61
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Eight out of ten proficiency chemicals listed in the guideline were tested successfully. The results confirmed the classification reported in the OECD guideline (LAUS reference study 201704R875).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:
other: study cannot be used for classification alone
Conclusions:
The test item shows only minimal reactivity towards cysteine and lysine containing peptides and therefore no potential to cause skin sensitization.
Executive summary:

This study was performed in order to estimate the skin sensitisation potential of the test item using a peptide model. The direct peptide reactivity assay (DPRA) is anin chemicoassay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential. To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC. The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible. In this assay, the test item as well as the positive control (2,3-Butanedione) and the solvent control were tested in triplicate. 2,3-Butanedione shows a midrange depletion for the lysine peptide. As a result for the test item, the mean peptide depletion percentage was 1.69 for the Cys-Pepetide and 0.61 for the Lys-Peptide. The mean percentage of depletion of both peptides was 1.15, therefore predicting only minimal reactivity towards cysteine and lysine containing peptides and no potential to cause skin sensitization based on this reactivity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The potential of the substance to cause skin sensitization is examined in a weight-of-evidence approach using to in vitro / in chemico studies:

 

DPRA:

This study was performed in order to estimate the skin sensitisation potential of the test item using a peptide model. The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential. To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC. The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS

Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible. In this assay, the test item as well as the positive control (2,3-Butanedione) and the solvent control were tested in triplicate. 2,3-Butanedione shows a midrange depletion for the lysine peptide. As a result for the test item, the mean peptide depletion percentage was 1.69 for the Cys-Pepetide and 0.61 for the Lys-Peptide. The mean percentage of deletion of both peptides was 1.15, therefore predicting only minimal reactivity towards cysteine and lysine containing peptides and no potential to cause skin sensitization based on this reactivity.

 

LuSens:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined. In the experiments, the highest nominal applied concentration (2000 μM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in none of the experiments. DMSO (final concentration: 1 %) was used as solvent control. Lactic acid (5000 μM) was used as negative control and EGDMA (120 μM) as positive control. No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal concentration of the test item. Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor.

 

As a conclusion of this weight-of evidence approach, the test item was considered to have no potential to cause skin sensitization.

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. As a result the substance is not considered to be classified as a skin sensitizer under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.