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Description of key information

Data in context of an integrated approach addressing three key events of the skin sensitisation Adverse Outcome Pathway (AOP) was generated with the test item TMA-Tempo. The data taken togehther revealed no skin sensitising potential of the test item.

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:
09.03.2018 to 14.08.2018
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
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
In order to reduce in vivo experiments, in chemico, in slico and/or in vitro methodes on skin sensitisation can be used. The DPRA test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”.
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 an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP.
Details on the study design:
In the present study TMA-TEMPO was dissolved in dist. water, based on the results of the pre-experiments to obtain a stock solution of 100mM.
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. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation.
Positive control results:
Phase separation was observed for the samples of the positive control including the co-elution control. Mean peptide depletion (Cystein): 70.09%; Mean peptide depletion (Lysine): 61.66%
Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50): Mean peptide depletion: 65.88% (high reactivity, sensitiser); Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10): Mean peptide depletion: 65.88% (moderate reactivity, sensitiser)
Key result
Run / experiment:
other: Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)
Parameter:
other: mean peptide depletion [%]
Value:
5.75
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: inconclusive
Run / experiment:
other: Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Parameter:
other: mean peptide depletion [%]
Value:
10.88
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: not applicable
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes - historical data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: reference control A, B, C pass the acceptance criteria for both peptides (Cysteine and Lysine)
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: met acceptance criteria for positive control and reference controls A, B, C for both peptides (Cysteine and Lysine)

According to the evaluation criteria in the guideline, for test items with combined cysteine / lysine peptide depletion between 3% and 10% a second run should be considered. Therefore, no prediction can be made.

Interpretation of results:
other: results can be used in an IATA to decide on classification or no classification for skin sensitisation
Remarks:
inconclusive
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both
peptides. Due to the evaluation criteria in the guideline the prediction model does not apply 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.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02.05.2018 to 10.07.2018
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
In the present study TMA-TEMPO was dissolved in dist. water. Based on a molecular weight of 249.8 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.
Key result
Run / experiment:
other: Luciferase Activity Experiment 1
Parameter:
other: Induction of Luciferase activity [maximal induction factor, I max]
Value:
102 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: Luciferase Activity Experiment 2
Parameter:
other: Induction of Luciferase activity [maximal induction factor, I max]
Value:
108 %
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In both experiments, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
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.
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:
key study
Study period:
14.02.2019 to 12.08.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 442E: (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: DB-ALM Protocol n°158: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation
Version / remarks:
July 1st, 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
In order to reduce in vivo experiments, in chemico, in slico and/or in vitro methodes on skin sensitisation can be used. The DPRA test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into UN GHS “Category 1”.
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 an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP.
Positive control results:
Positive control DNCB led to upregulation of CD86 and CD54 in all experiments.
Run / experiment:
other: #1
Parameter:
other: RFI CD86
Value:
150
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not upregulated
Remarks:
at any concentration
Run / experiment:
other: #2
Parameter:
other: RFI CD86
Value:
150
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: upregulated
Remarks:
at several concentrations
Run / experiment:
other: #3
Parameter:
other: RFI CD86
Value:
150
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not upregulated
Remarks:
at any concentration
Run / experiment:
other: #1
Parameter:
other: RFI CD54
Value:
200
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not upregulated
Remarks:
at any concentration
Run / experiment:
other: #2
Parameter:
other: RFI CD54
Value:
200
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not upregulated
Remarks:
at any concentration
Run / experiment:
other: #3
Parameter:
other: RFI CD54
Value:
200
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not upregulated
Remarks:
at any concentration

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps: 5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49, 1395.41 µg/mL.

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 91.7% (CD86), 91.7% (CD54) and 92.1% (isotype IgG1 control) in the first experiment, 95.6% (CD86), 94.9% (CD54) and 95.4% (isotype IgG1 control) in the second experiment and 96.0% (CD86), 95.6% (CD54) and 95.9% (isotype IgG1 control) in the third experiment.

All acceptance criteria were met for cell viability, RFI of solvent control (CD86 and CD54), RFI of positive control (CD86 and CD54), MFI ratios of CD86/CD54 to isotype IgG1.

The expression of the cell surface marker CD86 was above the threshold in several concentrations unpregulated up to 262% in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in the other two experiments in all concentrations.

The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments and concentrations.

Relative Fluorescence Intensity (RFI) of CD54 and CD86 in all experiments

Sample

Conc.
[μg/mL]

Experiment 1

Relative Fluorescence Intensity (RFI)

Experiment 2

Relative Fluorescence Intensity (RFI)

Experiment 3

Relative Fluorescence Intensity (RFI)

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

100

100

100

100

100

100

Solvent Control

0.20%

91

57

128

118

124

118

DNCB

4.0

261

252

560

318

187

445

TMA-TEMPO

5000.0

94

84

128

104

117

156

4166.67

87

65

181

114

122

194

3472.22

93

59

217

94

115

168

2893.52

84

61

122

85

121

187

2411.27

89

56

250

160

79

114

2009.39

78

56

262

139

95

158

1674.49

81

66

224

177

113

177

1395.41

104

59

233

145

111

166

Interpretation of results:
other: results can be used in an IATA to decide on classification or no classification for skin sensitisation
Remarks:
considered as non-sensitiser
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item can 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 conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Key event 1 - in chemico peptide/protein binding (DPRA, OECD TG 442C):

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. 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. The test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (5.75%). According to the evaluation criteria in the guideline, for test items with combined cysteine / lysine peptide depletion between 3% and 10% a second run should be considered.

In this study under the given conditions the test item showed minimal reactivity towards both peptides. Due to the evaluation criteria in the guideline (for test items with combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered) the prediction model does not apply and a final prdiction cannot be made.

Key event 2 - in vitro keratinocyte response (KeratinoSens, OECD TG 442D):

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. 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. 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. In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

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.

Key event 3 - in vitro dendritic cell response (h-CLAT, OECD TG 442E):

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The main experiment was performed covering the following concentration steps: 5000, 4166.67, 3472.22, 2893.52, 2411.27, 2009.39, 1674.49, 1395.41 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. No cytotoxic effects were observed for the cells treated with the test item at any test condition. The expression of the cell surface marker CD86 was upregulated up to 262% at a concentration of 2009.39 μg/mL in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in the other two experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Weight of evidence approach:

Considering all available information on skin senistisation testing covering the three key events for skin sensitisation an estimation on the skin sensitising potential of the test item TMA-Tempo could be made. Each test addressed a specific key event of the Adverse Outcome Pathway (AOP). The first key event of protein binding revealed an inconlussive result because only one experimental run was perfomed with mean peptide depletion of both peptides of 5.75%.

However, the mean depletion of both peptides in that single run was below the threshold of 6.38% for postive prediction. The result falls into the reactivity class of no to minimal reactivity in means of negative indication for skin sensitisation. The tests of the key events addressing keratinocyte and dendritic cell response were negative concerning skin sensitisation. All in all, two out of three tests gave clearly negative results on skin sensitisation which means classification of the test item TMA-Tempo as non-sensitiser with one supporting but borderline result indicating as well negative prediction for skin sensitisation. This supports the decision on no classification of TMA-Tempo for skin sensitisation.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the weight of evidence approach for the test item TMA-Tempo on skin sensitisation no classification according to CLP Regulation (EC) No. 1272/2008 is indicated.