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Description of key information

Thein chemicodirect 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 this study the test item showed minimal reactivity towards the peptides. Since the test item could not be tested with a 100 mM stock solution, the obtained negative results might not reflect the true reactivity and a prediction cannot be made.

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser.

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 this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.

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:
key study
Study period:
2017
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))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
This in chemico method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using high performance liquid chromatography (HPLC).

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.
Specific details on test material used for the study:
Batch No.: 80-47-16
Storage Conditions: Room temperature, protected from light
Expiry Date: August 2017
Details on the study design:
In the present study RED 2735 was dissolved in methanol at a concentration of 2.5 mg/mL based on the results of the pre-experiments. Since no molecular weight could be derived the test item was tested to its maximum solubility. 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 analysis.
Run / experiment:
other: Mean Peptide Depletion [%]
Parameter:
other: Depletion of the Cysteine Peptide
Value:
0
Vehicle controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Mean Peptide Depletion [%]
Parameter:
other: Depletion of the Lysine Peptide
Value:
3.36
Vehicle controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Mean Peptide Depletion [%]
Parameter:
other: Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50
Value:
1.68
Positive controls validity:
valid
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the peptides. Since the test item could not be tested with a 100 mM stock solution, the obtained negative results might not reflect the true reactivity and a prediction cannot be made.
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.
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 RED 2735 was dissolved in methanol at a concentration of 2.5mg/mL based on the results of the pre-experiments. Since no molecular weight could be derived the test item was tested to its maximum solubility. 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 analysis.

For the 2.5 mg/mL stock solution of the test item no precipitation, turbidity or phase separation was observed upon mixing of the test item and the cysteine peptide. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 2.5 mg/mL stock solution of the test item precipitation was observed upon mixing of the test item and the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected again for precipitation, turbidity or phase separation. Precipitation of the test item was not detected anymore. Turbidity was observed for the samples of the positive control including the co-elution control of the positive control. 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 precipitation and phase separation were regarded as insignificant.

As no co-elution of test item with the peptide peaks was observed and as the observed precipitation upon mixing of the test item and the lysine peptide solution was not detected anymore after the 24 h incubation period, sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 2.5 mg/mL stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤6.38% (1.68%). The test item would be categorized as non-sensitiser. However, since the test item was not tested with a 100 mM stock solution, the prediction might be not applicable.

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 64.30%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
This in vitro method is designed to predict and classify the skin sensitising potential of a chemical or multi-constituent substances or mixtures by assessment of its potential to upregulate cell surface markers using fluorescence-activated cell sorting (FACS).

The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.
Specific details on test material used for the study:
Batch No.: 80-47-16
Storage Conditions: room temperature, protected from light
Expiry Date: August 2017
Details on the study design:
In the present study RED 2735 was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 400 mg/mL to 3.13 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. No CV75 could be derived in the dose finding assay.
Based on that result, the main experiment was performed covering the following concentration steps: 800.0, 666.7, 555.6, 462.9, 385.8, 321.5, 267.9, 223.3 µg/mL
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
Run / experiment:
other: Experiment 1 - test item concentration range from 800.00 – 223.3 µg/mL - (CD86)
Parameter:
other: Relative Flourescence Intensity (RFI)
Value:
99
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2 - test item concentration range from 800.00 – 223.3 µg/mL - (CD86)
Parameter:
other: Relative Flourescence Intensity (RFI)
Value:
52
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 1 - test item concentration range from 800.00 – 223.3 µg/mL - (CD54)
Parameter:
other: Relative Flourescence Intensity (RFI)
Value:
88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2 - test item concentration range from 800.00 – 223.3 µg/mL- (CD54)
Parameter:
other: Relative Flourescence Intensity (RFI)
Value:
65
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Strong precipitation of the test item was observed after diluting the THF stock solutions with cell culture medium down to a concentration of 25 µg/mL. Furthermore, the precipitates stuck to the wall of the tubes.
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser. However, as the test item showed strong precipitates which stuck to the wall of the tubes down to a concentration of 25 μg/mL, it is questionable, if an entire test item contact to the cells at these concentrations occurred. Furthermore, as no cytotoxic effect was observed, there is no evidence that the cells had contact to the test item. Therefore, the result has to be considered with care.
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.
Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study RED 2735 was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 400 mg/mL to 3.13 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. No CV75 could be derived in the dose finding assay.

Based on that result, the main experiment was performed covering the following concentration steps: 800.0, 666.7, 555.6, 462.9, 385.8, 321.5, 267.9, 223.3 μg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Strong precipitation of the test item was observed after diluting the THF stock solutions with cell culture medium down to a concentration of 25 μg/mL. Furthermore, the precipitates stuck to the wall of the tubes.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 95.4% (CD86), 95.3% (CD54) and 95.2% (isotype IgG1 control) in the first experiment and to 97.7% (CD86), 97.2% (CD54) and 97.5% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered to be no skin sensitiser. In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin sensitiser. However, as the test item showed strong precipitates which stuck to the wall of the tubes down to a concentration of 25 μg/mL, it is questionable, if an entire test item contact to the cells at these concentrations occurred. Furthermore, as no cytotoxic effect was observed, there is no evidence that the cells had contact to the test item. Therefore, the result has to be considered with care.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (391% experiment 1; 462% experiment 2) and 200% for CD54 (327% experiment 1; 369% experiment 2) were clearly exceeded.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection.

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals
Specific details on test material used for the study:
Batch No.: 80-47-16
Storage Conditions: room temperature, protected from light
Expiry Date: August 2017
Details on the study design:
In the present study RED 2735 was dissolved in THF.
Since the test item had no defined molecular weight, the test was be performed using a pro forma molecular weight of 200 g/mol. Based on this, 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.
Run / experiment:
other: Experiment 1 - test item concentration of 125 µM
Parameter:
other: max luciferase activity (Imax) induction
Value:
3.21
Positive controls validity:
valid
Run / experiment:
other: Experiment 2 - test item concentration of 62.50 µM
Parameter:
other: max luciferase activity (Imax) induction
Value:
3.1
Positive controls validity:
valid
Run / experiment:
other: Mean value
Parameter:
other: max luciferase activity (Imax) induction
Value:
3.16
Positive controls validity:
valid
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.
Executive summary:

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 RED 2735 was dissolved in THF.

Since the test item had no defined molecular weight, the test was be performed using a pro forma molecular weight of 200 g/mol.Based on this, 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, a max luciferase activity (Imax) induction of 3.21 was determined at a test item concentration of 125 µM. At this concentration no cytotoxic effect was observed microscopically.The lowest tested concentration with a significant luciferase induction >1.5 (2.66) was found to be 62.50 µM.Microscopically, no cytotoxic effect was observed at this concentration. The calculated EC1.5was < 200 µg/mL (7.12 µg/mL corresponding to 35.60 µM).

In the second experiment, a max luciferase activity (Imax) induction of 3.10 was determined at a test item concentration of 62.50 µM. At this concentration no cytotoxic effect was observed microscopically.The lowest tested concentration with a significant luciferase induction >1.5 (1.89) was found to be 31.25 µM.Microscopically, no cytotoxic effect was observed at this concentration. The calculated EC1.5was < 200 µg/mL (4.25 µg/mL corresponding to 21.27 µM).

A 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 sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the integrated testing strategy adopted for the evaluation of the skin sensitization potential, Red 2735 is to be considered as a skin sensitizer and therefore it is classified as Skin Sens 1, H317.