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Description of key information

Skin sensitisation:

Direct Peptide Reactivity Assay (DPRA): considered as "non-sensitiser".

KeratinoSens study: considered as non-sensitiser.

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:
From 2018-04-24 to 2018-05-07
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
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
Batch No.: 20170707
Purity: 99.13%
Details on the study design:
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 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 h ± 1 h in assay medium at 37 °C ± 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 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

-Luciferase activity:
After 48 h ± 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 1.000 ms before assessing the luciferase activity for 2.000 ms. 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 °C ± 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 °C ± 1 °C and 5% CO2 over the weekend (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
Key result
Run / experiment:
other: first experiment
Parameter:
other: Imax
Value:
1.89
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item concentration is 2000 µM. The corresponding cell viability was 43.0%.
Key result
Run / experiment:
other: first experiment
Parameter:
other: EC1.5
Value:
613.76
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: second experiment
Parameter:
other: Imax
Value:
1.56
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The test item concentration is 1000 µM. The corresponding cell viability was 38.0%.
Key result
Run / experiment:
other: second experiment
Parameter:
other: EC1.5
Value:
853.66
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In the first experiment, a max luciferase activity (Imax) induction of 1.89 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 43.0%. The lowest tested concentration with a significant luciferase induction >1.5 (1.67) was found to be 1000 µM. The corresponding cell viability was <70% (48.8%). The calculated EC1.5was<1000 µM (613.76).
In the second experiment, a max luciferase activity (Imax) induction of 1.56 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 38.0%. This was the only tested concentration with a significant luciferase induction. The calculated EC1.5was<1000 µM (853.66).
No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
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:

The test was performed with OECD 442D, 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.

In the present study the test item was dissolved in DMSO. Based on a molecular weight of 146.15 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 and a stable suspension was formed. 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 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.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2018-02-16 to 2018-03-01
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:
other: Direct Peptide Reactivity Assay (DPRA)
Specific details on test material used for the study:
Batch No.: 20170707
Purity: 99.13%
Details on the study design:
TEST SYSTEM
- Preparation of the Test Item: The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.
- Controls: Reference controls, co-elution controls and a positive control (PC, Cinnamic aldehyde)
- HPLC System: Agilent Infinity 1260 II with Chromeleon 7.2 SR5
- Peptides: 19.65 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.10 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
19.84 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 (37.68 mL) to reach a concentration of 0.667 mM.

EXPERIMENTAL PROCEDURE
- Pre-Experiments: The test item was not soluble in acetonitrile and dist. water. The test item was completely soluble in dist. water: acetonitrile 1:1 (v/v).
- Incubation of the Test Item with the Cysteine and Lysine Peptide: 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). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides.
Positive control results:
Mean Peptide Depletion of the Cysteine Peptide: 77.79% ± 0.34%.
Mean Peptide Depletion of the Lysine Peptide: 66.10% ± 0.50%.
The acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.
Key result
Run / experiment:
other: Cysteine Peptide Depletion
Parameter:
other: Mean Peptide Depletion [%]
Value:
1.18
Positive controls validity:
valid
Key result
Run / experiment:
other: Lysine Peptide Depletion
Parameter:
other: Mean Peptide Depletion [%]
Value:
100
Positive controls validity:
valid
Other effects / acceptance of results:
For the 100 mM 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 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). No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.
A strong co-elution of the test item with the lysine peptide peak was observed. Therefore, the values of the peak areas of the lysine experiment cannot be used for evaluation.
Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item might be considered as “non-sensitiser”.
Executive summary:

The sensitising potential of test item was detected by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteinein chemicodirect peptide reactivity assay (DPRA) according to OECD442C.

In the present study test item was dissolved in water : acetonitrile 1:1 (v/v), based on the results of the pre-experiments. Based on a molecular weight of 146.15 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 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, no precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM 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, phase separation was observed for the samples of the positive control (including the co-elution control). No precipitation, turbidity or phase separation was observed for 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 insignificant.

A strong co-elution of the test item with the lysine peptide peak was observed. Therefore, the values of the peak areas of the lysine experiment cannot be used for evaluation.

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 C (RC C dist. water : acetonitrile 1:1 (v/v)).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was≤ 13.89% (1.18%). Based on the prediction model 2 the test item can be considered as non-sensitiser.

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

Skin sensitisation:

In the Direct Peptide Reactivity Assay (DPRA), the test item showed minimal reactivity towards both peptides. The test item might be considered as "non-sensitiser".

In the KeratinoSens study 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.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Two in vitro studies (DPRA, KeratinoSens) howed this substance is non-sensitiser.

According to Regulation (EC) No 1272/2008, table 3.4.2, this substance is not classified for this endpoint.