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Diss Factsheets

Administrative data

Description of key information

DPRA: Under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might be considered as "non-sensitiser" (reference 7.4.1-1).

KeratinoSens: Under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser (reference 7.4.1-2).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2018-12-05 to 2019-02-06
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))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
Deviations:
no
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) - Details on study design:

- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; >95 %; Lot. No.: 111016HS_MHeW0318
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; >95 %; Lot. No.: 120514HSDW_W0318

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls: cysteine or lysine peptide in acetonitrile with and without test item

Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.

Peptide stock solution preparation:
- 20.69 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.36 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 18.97 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (35.13 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). Three samples of the test substance in acetonitrile were incubated with each peptide for 24 ± 2 h at room temperature (25 ± 2.5 °C) in the dark. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

HPLC conditions:
HPLC/DAD: Agilent Infinity 1260 II with Chromeleon 7.2 SR5
Detection: 220 nm signal for quantitation, 258 nm signal used as indicator for co-elution
Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 μ m, Agilent Art. Nr. 861753-902
Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm.
Column Temperature: 30 °C
Sample Temperature: 20 - 25 °C
Run Time: 20 minutes

Gradient:
Time Flow %A %B
0 min 0.35 mL/min 90 10
10 min 0.35 mL/min 75 25
11 min 0.35 mL/min 10 90
13 min 0.35 mL/min 10 90
13.5 min 0.35 mL/min 90 10
20 min end run

Injection Volume: 4 μL
HPLC Mobile Phase A: 0.1 % ( v/v) trifluoroacetic acid in water
HPLC Mobile Phase B: 0.085 % ( v/v) trifluoroacetic acid in acetonitrile

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at A = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C)) * 100

Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r2 > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8 % and 100 % for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9 %,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2 % and 69.0 % for the lysine peptide and the maximum SD for the positive control replicat es is < 11.6 %,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0 %.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9 % for the cysteine per cent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6 % for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Evaluation of results:
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38 % average peptide depletion was used to support the discrimination between skin sensitisers and nonsensitisers. By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89 % peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.
Positive control results:
The positive control caused depletion of both peptides comparable to historical data.
Key result
Run / experiment:
mean
Parameter:
other: combined peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
mean
Parameter:
other: cysteine peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
mean
Parameter:
other: lysine peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No precipitation or turbidity was observed for the samples of the test item. No phase separation with the cysteine peptide solution was observed. Slight phase separation with the lysine peptide solution was observed for all the samples of the test item. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.
- Co-elution: No relevant co-elution of the test item with any of the peptide peaks was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might be considered as "non-sensitiser".
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. In the present study, performed according to OECD 442C, the test item was given into acetonitrile, based on the results of the pre-experiments. The test item was completely soluble and the resulting solution was used for further testing. Based on a molecular weight of 146.11 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control. Slight phase separation was also observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of test item with the peptide peaks was observed. Slight phase separation in the lysine experiment was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 13.89 % (0.00 %). Based on the prediction model 2 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86 %.

Under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might be considered as “non-sensitiser”. 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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2018-12-05 to 2019-04-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, March 09, 2018
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
Skin sensitisation (In vitro test system)
Cell line used: KeratinoSens™ (Givaudan, Switzerland)

Technical material and conditions:
- Maintenance Medium: Dulbecco’s Modified Eagle Medium D-MEM (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate + 10 % fetal bovine calf serum FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM (GlutaMAX™) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate + 10 % FBS
- Test Item Exposure Medium: D-MEM with 1.0 g/L D-glucose and 1 mM Na-Pyruvate + 1 % FBS
- Luciferace reagent: 10 vials Luciferase Assay Substrate (lyophilized) (Promega, Cat. No.: E1501)
- Assay Buffer: 10 x 10 mL Luciferase Assay Buffer (Promega, Cat. No.: E1501)
- Lysisbuffer: 30 mL Luciferase Cell Culture Lysis 5x reagent (Promega Cat. No.: E1531), diluted 1:5 prior to the study
- MTT Solution: MTT stock solution: 5 mg/mL in DPBS
- SDS solution: 10% (w/v) sodium dodecyl sulfate 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: blank well without seeded cells

Test procedure:
A cell suspension of 8E4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1E4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 ± 1 h in assay medium at 37 ± 1 °C and 5 % CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 ± 1 h at 37 ± 1 °C and 5 % CO2.

Luciferase activity
After 48 ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 s before assessing the luciferase activity for 2 s. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 ± 1 °C and 5 % CO2. Afterwards the medium was removed and replaced by 200 μL 10 % SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 ± 1 °C and 5 % CO2 over the weekend. After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 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 test meets the acceptance criteria, if:
- 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.
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.45 in experiment 1; 4.41 in experiment 2). Therefore, the positive control fullfilled the validity criteria of the test.
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 (µM)
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 (µM)
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.37
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



Table 1: Results of the Cytotoxicity Measurement









































 



Concentration [μM]



Cell Viability [%]



Experiment 1



Experiment 2



Mean



SD



Solvent Control



-



100



100



100



0.0



Positive Control



4.00


8.00


16.00


32.00


64.00



95.8


106.7


94.1


78.5


74.8



88.1


97.1


94.3


95.0


94.4



92.0


101.9


94.2


86.8


84.6



5.4


6.8


0.2


11.7


13.8



Test Item



0.98


1.95


3.91


7.81


15.63


31.25


62.50


125.00


250.00


500.00


1000.00


2000.00



78.7


116.8


115.1


99.3


113.5


103.2


104.7


106.9


96.8


94.5


96.9


0.6



107.9


103.1


96.7


91.0


95.6


92.2


85.5


92.4


87.7


86.7


31.2


0.0



93.3


110.0


105.9


95.1


104.6


97.7


95.1


99.6


92.2


90.6


64.1


0.3



20.7


9.6


13.0


5.8


12.7


7.8


13.5


10.2


6.5


5.5


46.5


0.4



 


Table 2: Induction of Luciferase Activity Experiment 1

















































Experiment 1



Concentration [µM]



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


8.00


16.00


32.00


64.00



1.28


1.35


1.66


1.93


3.35



1.17


1.37


1.44


2.24


3.82



1.25


1.32


1.63


2.07


3.18



1.23


1.35


1.58


2.08


3.45



0.06


0.03


0.12


0.16


0.33



 


 


*


*


*



Test Item



0.98


1.95


3.91


7.81


15.63


31.25


62.50


125.00


250.00


500.00


1000.00


2000.00



0.92


0.95


1.08


0.85


1.06


1.01


1.24


1.37


1.19


1.45


1.28


0.05



1.15


0.96


0.84


0.82


0.95


1.11


1.17


1.10


1.19


1.37


1.08


1.09



0.83


0.78


0.91


0.87


0.90


0.93


0.95


1.13


1.22


1.29


1.07


0.76



0.96


0.90


0.94


0.85


0.97


1.02


1.12


1.20


1.20


1.37


1.14


0.63



0.17


0.10


0.13


0.02


0.08


0.09


0.15


0.15


0.02


0.08


0.12


0.53



 



* = significant induction according to Student’s t-test, p<0.05






Table 3: Induction of Luciferase Activity Experiment 2

















































Experiment 2



Concentration [µM]



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


8.00


16.00


32.00


64.00



1.21


1.35


1.46


2.50


4.20



1.26


1.38


1.62


1.95


4.36



1.13


1.20


1.43


1.93


4.66



1.20


1.31


1.50


2.13


4.41



0.06


0.10


0.10


0.32


0.23



 


 


*


*


*



Test Item



0.98


1.95


3.91


7.81


15.63


31.25


62.50


125.00


250.00


500.00


1000.00


2000.00



0.98


0.82


0.86


0.97


0.99


1.07


1.09


1.03


1.02


1.07


1.51


0.00



1.20


1.02


1.07


1.17


1.04


1.11


1.01


0.99


1.06


1.07


1.33


0.00



1.04


0.96


0.95


0.97


1.17


0.95


1.08


1.00


0.96


0.98


1.06


0.02



1.07


0.93


0.96


1.04


1.07


1.04


1.06


1.00


1.02


1.04


1.30


0.01



0.11


0.10


0.10


0.12


0.09


0.08


0.04


0.02


0.05


0.06


0.23


0.01



 



* = significant induction according to Student’s t-test, p<0.05


Table 4: Induction of Luciferase Activity – Overall Induction














































 



Concentration [µM]



Fold Induction



Significance



Experiment 1



Experiment 2



Mean



SD



 



Solvent Control



-



1.00



1.00



1.00



0.00



 



Positive Control



4.00


8.00


16.00


32.00


64.00



1.23


1.35


1.58


2.08


3.45



1.20


1.31


1.50


2.13


4.41



1.21


1.33


1.54


2.10


3.93



0.02


0.03


0.05


0.03


0.67



 


 


*


*


*



Test Item



0.98


1.95


3.91


7.81


15.63


31.25


62.50


125.00


250.00


500.00


1000.00


2000.00



0.96


0.90


0.94


0.85


0.97


1.02


1.12


1.20


1.20


1.37


1.14


0.63



1.07


0.93


0.96


1.04


1.07


1.04


1.06


1.00


1.02


1.04


1.30


0.01



1.02


0.91


0.95


0.94


1.02


1.03


1.09


1.10


1.11


1.21


1.22


0.32



0.08


0.03


0.01


0.13


0.07


0.02


0.04


0.14


0.13


0.23


0.11


0.44



 



* = significant induction according to Student’s t-test, p<0.05


Table 5: Additional Parameters









































Parameter



Experiment 1



Experiment 2



Mean



SD



EC1.5 [μM]



n.a.



n.a.



n.a.



n.a.



Imax



1.37



1.30



1.33



0.05



IC30 [µM]



1279.53



650.11



964.82



445.07



IC50 [µM]



1487.16



830.38



1158.77



464.41



n.a. = not applicable


Table 6: Acceptance Criteria














































Criterion



Range



Experiment 1



pass/fail



Experiment 2



pass/fail



CV Solvent Control



< 20 %



11.9



pass



5.1



pass



No. of positive control concentration steps with significant luciferase activity induction >1.5



≥ 1



3.0



pass



3.0



pass



EC1.5 PC



± 2xSD of historical mean



13.25



pass



15.84



pass



Induction PC at 64 µM



2.00 < x < 8.00



3.45



pass



4.41



pass



 

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
Executive summary:



A study according to OECD 442D was performed to predict the skin sensitising potential of the test item in the in vitro KeratinoSens™ assay. In the present study the test item was dissolved in DMSO. Based on a molecular weight of 146.11 g/mol a stock solution of 200 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: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 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, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. The test item was cytotoxic at the highest concentration.
In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. The test item was cytotoxic at the two highest concentrations.
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non-sensitiser. The controls confirmed the validity of the study

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

OECD 442C - DPRA

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. In the present study, performed according to OECD 442C, the test item was given into acetonitrile, based on the results of the pre-experiments. The test item was completely soluble and the resulting solution was used for further testing. Based on a molecular weight of 146.11 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control including the co-elution control. Slight phase separation was also observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of test item with the peptide peaks was observed. Slight phase separation in the lysine experiment was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 13.89 % (0.00 %). Based on the prediction model 2 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86 %.

Under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might be considered as “non-sensitiser”. 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.

OECD 442D - KeratinoSens

A study according to OECD 442D was performed to predict the skin sensitising potential of the test item in the in vitro KeratinoSens™ assay. In the present study the test item was dissolved in DMSO. Based on a molecular weight of 146.11 g/mol a stock solution of 200 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: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 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, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. The test item was cytotoxic at the highest concentration.
In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. The test item was cytotoxic at the two highest concentrations.
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non-sensitiser. The controls confirmed the validity of the study.

Conclusion

Based on the results obtained in the two in vitro tests, the test item showed no skin sensitising potential and therefore, is not considered to be a skin sensitiser.

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 twelfth time in Regulation (EU) No. 2019/521.