Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Description of key information
This endpoint was assessed in a weight of evidence approach.
Three key elements of the AOP for skin sensitisation have been adressed in vitro. The results are shown below:
OECD 442 C: inconclusive due to precipitation of the test material
OECD 442 D: non-sensitizer
OECD 442 E: non-sensitizer
Test item showed minimal reactivity towards the cysteine peptide in the OECD Guideline 442C. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made. However, the test item does not activate keratinocytes (OECD 442D) and did not upregulate the expression of the cell surface markers (OECD 442E). Therefore, it can be concluded that the test item is not sensitizing to skin.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-10-19 to 2017-11-25
- 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
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
- Version / remarks:
- 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- 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.
- Positive control results:
- 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 64.04%.
- Key result
- Run / experiment:
- other: cysteine run
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: lysine run
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- 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
replicates 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
percent 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.
Both peptide runs and the test item results met the acceptance criteria of the test. - Interpretation of results:
- study cannot be used for classification
- Conclusions:
- In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP. - Executive summary:
In the present study the test material was dissolved in acetonitrile, based on the results of the pre-experiments
Based on a molecular weight of 338 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. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control). Phase separation was observed for the samples of the test item. Samples of the positive control were centrifuged prior to the HPLC analysis.
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 (excluding the co-elution control of the positive control). Phase separation was also observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.
Since the acceptance criteria of the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.
Due to the observed phase separation after the incubation period in the lysine peptide samples, prediction model 2 based on the cysteine peptide depletion only should be considered. However, precipitation was observed as well in the cysteine experiment.
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was ≤ 6.38 % (0 %).
According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.
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 64.04 %.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-12-15 to 2018-04-30
- 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:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
- Version / remarks:
- 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- activation of keratinocytes
- Details on the study design:
- 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.
- Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.26 (experiment 1); 4.34 (experiment 2)).
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Value:
- 1.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 15.63 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity
- Value:
- 1.11
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 15.63 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 90.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: at 15.63 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 81.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: at 15.63 µM
- Other effects / acceptance of results:
- Acceptance Criteria
The test meets 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.
The controls fullfilled the validity criteria of the test. - 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.
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:
In the present study the test material was dissolved in DMSO. Based on a molecular weight of 338 g/mol a stock solution of 200 mM was prepared.The main experiments were performed using two different batches (experiment 1: D017011497; experiment 2: D017144397) because of the expiry date of the first batch of the test item.
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.
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.
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.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-04-30 to 2018-07-17
- 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 (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- activation of dendritic cells
- Details on the study design:
- 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.
- Positive control results:
- The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both
experiments. The threshold of 150 % for CD86 (273 % experiment 1; 348 % experiment 2) and
200 % for CD54 (250 % experiment 1; 371 % experiment 2) were clearly exceeded. - Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 449
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 51.06 µg/mL
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 678
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 42.55 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 282
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 51.06 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 337
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 51.06 µg/mL
- Other effects / acceptance of results:
- Acceptance criteria:
The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90 %,
• the cell viability of at least four tested doses of the test item in each run is >50 %,
• the RFI values of the positive control (DNCB) is ≥150 % for CD86 and ≥200 % for CD54 at a cell viability of >50 %,
• the RFI values of the solvent control is not ≥150 % for CD86 and not ≥200 % for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105 %.
The test mets the acceptance criteria. - Interpretation of results:
- GHS criteria not met
- 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 is 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. - Executive summary:
In the present study the test material was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 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. In the dose finding assay, precipitates and turbidity and oily droplets were observed for working solutions 1 – 4 (500 mg/mL – 62.5 mg/ml - 1000 µg/ml – 125 µg/mL applied concentration) when diluted 1:250 in cell culture medium.
A CV75 of 42.55 ± 19.77 µg/mL was derived in the dose finding assay.
Based on the CV75, the main experiment was performed covering the following concentration steps:
51.06, 42.55, 35.46, 29.55, 24.63, 20.52, 17.10, 14.25 µg/mL
In the main experiment no precipitation or turbidity of the test item was observed for all tested concentration steps when diluted 1:250 in cell culture medium.
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.
Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 9.2 % (CD86), 8.8 % (CD54) and 8.7 % (isotype IgG1 control) in the first experiment and to 5.4 % (CD86), 4.6 % (CD54) and 4.4 % (isotype IgG1 control) in the second experiment.
The expression of the cell surface marker CD86 was upregulated to 449 % and 389 % in the first experiment and to 282 % in the second
experiment. The upregulation above the threshold of 150 % was observed at a concentration of 51.06 µg/mL and 42.55 µg/mL in the first experiment and at a concentration of 51.06 µg/mL in the second experiment.The expression of the cell surface marker CD54 was upregulated to 626 %, 678 % and 263 % in the first experiment and to 337 % in the second experiment. The upregulation above the threshold of 200 % was observed at a concentration of 51.06 µg/mL, 42.55 µg/mL and 35.46 µg/mL in the first experiment and at a concentration of 51.06 µg/mL in the second experiment.
The induction of the expression of both cell surface markers occurred only at cytotoxic concentrations. Since there was no induction of the expression of the cell surface markers CD86 and CD54 in the other non-cytotoxic concentrations, the test item can be considered as non-sensitiser.
Referenceopen allclose all
Cysteine and Lysine Values of the Calibration Curve
Sample | Cysteine Peptide | Lysine Peptide | ||
Peak Area | Peptide Concentration [mM] | Peak Area | Peptide Concentration [mM] | |
STD1 | 4523.9644 | 0.5340 | 4455.4287 | 0.5340 |
STD2 | 2255.1311 | 0.2670 | 2248.6604 | 0.2670 |
STD3 | 1081.2599 | 0.1335 | 1103.8511 | 0.1335 |
STD4 | 538.3588 | 0.0667 | 555.1791 | 0.0667 |
STD5 | 264.6839 | 0.0334 | 278.7628 | 0.0334 |
STD6 | 131.3584 | 0.0167 | 141.0930 | 0.0167 |
STD7 | 0.0000 | 0.0000 | 0.0000 | 0.0000 |
Depletion of the Cysteine Peptide
Cysteine Peptide | ||||||
Sample | Peak Area | Peptide Conc. [mM] | Peptide Depletion [%] | Mean Peptide Depletion [%] | SD of Peptide Depletion [%] | CV of Peptide Depletion [%] |
Positive Control | 1320.4471 | 0.1577 | 70.95 | 71.10 | 0.18 | 0.25 |
1315.4908 | 0.1571 | 71.06 | ||||
1304.7782 | 0.1559 | 71.29 | ||||
Test Item | 4595.2119 | 0.5431 | 0.00 | 0.00 | 0.00 | n/a |
4572.8872 | 0.5405 | 0.00 | ||||
4551.4190 | 0.5379 | 0.00 | ||||
Depletion of the Lysine Peptide
Lysine Peptide | ||||||
Sample | Peak Area | Peptide Conc. [mM] | Peptide Depletion [%] | Mean Peptide Depletion [%] | SD of Peptide Depletion [%] | CV of Peptide Depletion [%] |
Positive Control | 1768.9691 | 0.2117 | 57.97 | 56.98 | 1.07 | 1.87 |
1804.7944 | 0.2160 | 57.12 | ||||
1858.1759 | 0.2224 | 55.85 | ||||
Test Item | 4243.7490 | 0.5080 | 0.00 | 0.00 | 0.00 | n/a |
4209.2564 | 0.5038 | 0.00 | ||||
4209.8970 | 0.5039 | 0.00 | ||||
Prediction Model 1
Cysteine 1:10/ Lysine 1:50 Prediction Model 1
Mean Cysteine andLysine PPD | Reactivity Class | DPRA Prediction² |
0.00 % ≤ PPD ≤ 6.38 % | No or Minimal Reactivity | Negative |
6.38 % < PPD ≤ 22.62 % | Low Reactivity | Positive |
22.62 % < PPD ≤ 42.47 % | Moderate Reactivity | |
42.47 % < PPD ≤ 100 % | High Reactivity |
1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.
2 DPRA predictions should be considered in the framework of an IATA.
Prediction Model 2
Cysteine 1:10 Prediction Model
Cysteine PPD | ReactivityClass | DPRA Prediction² |
0.00 % ≤ PPD ≤ 13.89 % | No or Minimal Reactivity | Negative |
13.89 % < PPD ≤ 23.09 % | Low Reactivity | Positive |
23.09 % < PPD ≤ 98.24 % | Moderate Reactivity | |
98.24 % < PPD ≤ 100 % | High Reactivity |
Categorization of the Test Item
Prediction Model | Prediction Model 1 | Prediction Model 2 | ||||
Test Substance | Mean Peptide Depletion [%] | Reactivity Category | Prediction | Mean Peptide Depletion [%] | Reactivity Category | Prediction |
Test Item | 0.00 | Minimal Reactivity | no sensitiser | 0.00 | Minimal Reactivity | no sensitizer |
Positive Control | 64.04 | High Reactivity | sensitizer | 71.10 | Moderate Reactivity | sensitizer |
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 | 102.8 | 101.3 | 102.1 | 1.1 |
8.00 | 113.1 | 100.5 | 106.8 | 8.9 | |
16.00 | 113.5 | 98.8 | 106.2 | 10.4 | |
32.00 | 125.3 | 101.8 | 113.5 | 16.6 | |
64.00 | 131.2 | 96.8 | 114.0 | 24.3 | |
Test Item | 0.98 | 102.9 | 97.6 | 100.3 | 3.7 |
1.95 | 97.2 | 102.0 | 99.6 | 3.4 | |
3.91 | 96.9 | 99.6 | 98.3 | 1.9 | |
7.81 | 94.9 | 94.4 | 94.6 | 0.4 | |
15.63 | 90.7 | 81.4 | 86.1 | 6.5 | |
31.25 | 22.9 | 15.9 | 19.4 | 5.0 | |
62.50 | 0.0 | 0.0 | 0.0 | 0.0 | |
125.00 | 0.0 | -0.1 | -0.1 | 0.1 | |
250.00 | 0.1 | -0.1 | 0.0 | 0.1 | |
500.00 | 0.2 | 0.0 | 0.1 | 0.1 | |
1000.00 | 0.5 | 0.0 | 0.2 | 0.3 | |
2000.00 | 0.5 | 0.1 | 0.3 | 0.3 | |
Induction of Luciferase Activity Experiment 1 (Batch D017011497)
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 | 1.13 | 1.14 | 1.12 | 1.13 | 0.01 |
|
8.00 | 1.20 | 1.12 | 1.07 | 1.13 | 0.07 |
| |
16.00 | 1.33 | 1.61 | 1.23 | 1.39 | 0.19 |
| |
32.00 | 2.10 | 1.76 | 1.72 | 1.86 | 0.21 | * | |
64.00 | 4.82 | 4.21 | 3.76 | 4.26 | 0.53 | * | |
Test Item | (0.98 | 1.10 | 1.06 | [2.46] | 1.54 | 0.80) |
|
1.95 | 1.16 | 0.99 | 0.95 | 1.03 | 0.11 |
| |
3.91 | 1.07 | 1.17 | 0.99 | 1.08 | 0.09 |
| |
7.81 | 1.22 | 1.06 | 1.15 | 1.14 | 0.08 |
| |
15.63 | 1.81 | 1.34 | 1.34 | 1.50 | 0.27 |
| |
31.25 | 0.70 | 1.38 | 1.30 | 1.13 | 0.37 |
| |
62.50 | 0.00 | 0.68 | 0.24 | 0.31 | 0.34 |
| |
125.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
250.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
500.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
1000.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
2000.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
* = significant induction according to Student’s t-test, p<0.05
[outlier] according to statistical tests of Grubbs, Nalimov and Dixon
( ) concentration will not be used for evaluation
Induction of Luciferase Activity Experiment 2 (D017144397)
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 | 1.14 | 1.06 | 0.99 | 1.07 | 0.07 |
|
8.00 | 1.20 | 1.39 | 1.54 | 1.37 | 0.17 |
| |
16.00 | 1.28 | 1.59 | 1.57 | 1.48 | 0.18 |
| |
32.00 | 2.36 | 2.74 | 2.58 | 2.56 | 0.19 | * | |
64.00 | 4.04 | 5.02 | 3.97 | 4.34 | 0.58 | * | |
Test Item | 0.98 | 0.99 | 1.06 | 1.08 | 1.04 | 0.05 |
|
1.95 | 0.95 | 1.02 | 0.95 | 0.98 | 0.04 |
| |
3.91 | 0.97 | 0.97 | 1.02 | 0.99 | 0.03 |
| |
7.81 | 1.04 | 1.05 | 1.13 | 1.07 | 0.05 |
| |
15.63 | 1.19 | 1.04 | 1.11 | 1.11 | 0.08 |
| |
31.25 | 0.70 | 0.85 | 0.82 | 0.79 | 0.08 |
| |
62.50 | 0.55 | 0.77 | 0.71 | 0.68 | 0.11 |
| |
125.00 | 0.00 | 0.35 | 0.14 | 0.16 | 0.18 |
| |
250.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
500.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
1000.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
2000.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
* = significant induction according to Student’s t-test, p<0.05
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 | 1.13 | 1.07 | 1.10 | 0.05 |
|
8.00 | 1.13 | 1.37 | 1.25 | 0.17 |
| |
16.00 | 1.39 | 1.48 | 1.44 | 0.07 |
| |
32.00 | 1.86 | 2.56 | 2.21 | 0.50 |
| |
64.00 | 4.26 | 4.34 | 4.30 | 0.06 | * | |
Test Item | (0.98 | 1.54 | 1.04 | 1.29 | 0.35) |
|
1.95 | 1.03 | 0.98 | 1.00 | 0.04 |
| |
3.91 | 1.08 | 0.99 | 1.03 | 0.06 |
| |
7.81 | 1.14 | 1.07 | 1.11 | 0.05 |
| |
15.63 | 1.50 | 1.11 | 1.30 | 0.27 |
| |
31.25 | 1.13 | 0.79 | 0.96 | 0.24 |
| |
62.50 | 0.31 | 0.68 | 0.49 | 0.26 |
| |
125.00 | 0.00 | 0.16 | 0.08 | 0.11 |
| |
250.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
500.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
1000.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
2000.00 | 0.00 | 0.00 | 0.00 | 0.00 |
| |
* = significant induction according to Student’s t-test, p<0.05
( ) concentration will not be used for evaluation
Additional Parameters
Parameter | Experiment 1 | Experiment 2 | Mean | SD |
EC1.5[µM] | n.a. | n.a. | n.a. | n.a. |
Imax | 1.50 | 1.11 | 1.30 | 0.27 |
IC30[µM] | 20.39 | 18.35 | 19.37 | 1.44 |
IC50[µM] | 25.00 | 23.12 | 24.06 | 1.33 |
n.a.: not applicable
Acceptance Criteria
Criterion | Range | Experiment 1 | pass/fail | Experiment 2 | pass/fail |
CV Solvent Control | < 20 % | 7.8 | pass | 7.0 | pass |
No. of positive control concentration steps with significant luciferase activity induction >1.5 | ≥ 1 | 2.0 | pass | 2.0 | pass |
EC1.5 PC | 7 < x < 34 µM | 19.77 | pass | 16.27 | pass |
Induction PC at 64 µM | 2.00 < x < 8.00 | 4.26 | pass | 4.34 | pass |
Historical Data
Acceptance Criterion | Range | Mean | SD | N |
CV Solvent Control | < 20 % | 11.3 | 3.3 | 41 |
No. of positive control concentration steps with significant luciferase activity induction >1.5 | ≥ 1 | 2.3 | 0.6 | 41 |
EC1.5 PC | 7 < x < 34 µM | 20.4 | 6.7 | 41 |
Induction PC at 64 µM | 2.00 < x < 8.00 | 3.3 | 1.1 | 41 |
Results of the Cell Batch Activation Test (Batch 20)
Sample | Concentration | CD86 | CD54 | Activated | Pass /Fail | ||||
Cell Viability [%] | RFI | Threshold OECD TG 442E | Cell Viability [%] | RFI | Threshold OECD TG 442E | yes/no | |||
DNCB | 4 µg/mL | 89.2 | 266 | >150 | 88.4 | 206 | >200 | yes | pass |
NiSO4 | 100 µg/mL | 82.3 | 220 | >150 | 80.6 | 283 | >200 | yes | pass |
LA | 1000 µg/mL | 96.2 | 79 | ≤150 | 96.9 | 101 | ≤200 | no | pass |
Results of the Cell Batch Activation Test (Batch 21)
Sample | Concentration | CD86 | CD54 | Activated | Pass /Fail | ||||
Cell Viability [%] | RFI | Threshold OECD TG 442E | Cell Viability [%] | RFI | Threshold OECD TG 442E | yes/no | |||
DNCB | 4 µg/mL | 85.5 | 251 | >150 | 84.7 | 287 | >200 | yes | pass |
NiSO4 | 100 µg/mL | 74.9 | 249 | >150 | 75.5 | 518 | >200 | yes | pass |
LA | 1000 µg/mL | 94.8 | 67 | ≤150 | 95.0 | 108 | ≤200 | no | pass |
Results of the Dose Finding Assay
Sample | Experiment 1 | Experiment 2 | Experiment 3 | ||||
Concentration applied [µg/mL] | Cell Viability [%] | Concentration applied [µg/mL] | Cell Viability [%] | Concentration applied [µg/mL] | Cell Viability [%] | ||
Medium Control | -- | -- | 94.30 | -- | 93.90 | -- | 95.20 |
Solvent Control | DMSO | -- | 95.00 | -- | 92.90 | -- | 95.30 |
Test item | C8 | 7.81 | 94.40 | 7.81 | 91.30 | 7.81 | 94.60 |
C7 | 15.63 | 93.00 | 15.63 | 89.20 | 15.63 | 93.90 | |
C6 | 31.25 | 86.80 | 31.25 | 79.50 | 31.25 | 72.90 | |
C5 | 62.50 | 79.20 | 62.50 | 29.10 | 62.50 | 4.50 | |
C4 | 125.00 | 11.80 | 125.00 | 7.90 | 125.00 | 5.40 | |
C3 | 250.00 | 7.30 | 250.00 | 4.30 | 250.00 | 5.90 | |
C2 | 500.00 | 6.50 | 500.00 | 6.60 | 500.00 | 7.90 | |
C1 | 1000.00 | 14.10 | 1000.00 | 9.70 | 1000.00 | 11.90 | |
Calculated CV75 [µg/mL] | 65.26 | 33.25 | 29.16 | ||||
Mean CV75 [µg/mL] | 42.55 | ||||||
SD CV 75 [µg/mL] | 19.77 | ||||||
CD54 and CD86 Expression Experiment 1
Sample | Conc. | Cell Viability [%] | Mean Fluorescence Intensity | corrected Mean Fluorescence Intensity | Relative Flourescence Intensity (RFI) | Ratio Isotype IgG1 to [%] | |||||||
CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | CD86 | CD54 | CD86 | CD54 | ||
Medium Control | - | 94.5 | 93.7 | 94.2 | 3327 | 1407 | 668 | 2659 | 739 | 97 | 102 | 498 | 211 |
Solvent Control | 0.20 % | 94.5 | 93.2 | 93.9 | 3430 | 1410 | 686 | 2744 | 724 | 100 | 100 | 500 | 206 |
DNCB | 4.00 | 79.5 | 79.3 | 79.8 | 8105 | 2427 | 619 | 7486 | 1808 | 273 | 250 | 1309 | 392 |
Test item | 51.06 | 9.2 | 8.8 | 8.7 | 13774 | 5991 | 1461 | 12313 | 4530 | 449 | 626 | 943 | 410 |
42.55 | 9.1 | 9.4 | 9.0 | 12131 | 6367 | 1457 | 10674 | 4910 | 389 | 678 | 833 | 437 | |
35.46 | 24.1 | 23.3 | 22.4 | 4265 | 2753 | 849 | 3416 | 1904 | 124 | 263 | 502 | 324 | |
29.55 | 47.5 | 47.6 | 48.4 | 4093 | 1894 | 886 | 3207 | 1008 | 117 | 139 | 462 | 214 | |
24.62 | 75.1 | 73.8 | 74.0 | 3763 | 1686 | 744 | 3019 | 942 | 110 | 130 | 506 | 227 | |
20.52 | 84.2 | 83.7 | 84.1 | 3418 | 1612 | 692 | 2726 | 920 | 99 | 127 | 494 | 233 | |
17.10 | 88.2 | 88.7 | 87.6 | 3337 | 1572 | 685 | 2652 | 887 | 97 | 123 | 487 | 229 | |
14.25 | 90.6 | 90.8 | 90.6 | 3337 | 1502 | 693 | 2644 | 809 | 96 | 112 | 482 | 217 | |
CD54 and CD86 Expression Experiment 2
Sample | Conc. | Cell Viability [%] | Mean Fluorescence Intensity | corrected Mean Fluorescence Intensity | Relative Flourescence Intensity (RFI) | Ratio Isotype IgG1 to [%] | |||||||
CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | Isotype IgG1 | CD86 | CD54 | CD86 | CD54 | C86 | CD54 | ||
Medium Control | - | 97.2 | 97.0 | 96.7 | 2751 | 1282 | 649 | 2102 | 633 | 100 | 100 | 424 | 198 |
Solvent Control | 0.20 % | 97.1 | 97.0 | 97.0 | 2713 | 1251 | 618 | 2095 | 633 | 100 | 100 | 439 | 202 |
DNCB | 4.0 | 84.2 | 83.7 | 83.2 | 7946 | 3000 | 651 | 7295 | 2349 | 348 | 371 | 1221 | 461 |
Test item | 51.06 | 5.4 | 4.6 | 4.4 | 7306 | 3532 | 1398 | 5908 | 2134 | 282 | 337 | 523 | 253 |
42.55 | 30.3 | 30.1 | 29.5 | 3654 | 1796 | 704 | 2950 | 1092 | 141 | 173 | 519 | 255 | |
35.46 | 53.4 | 54.6 | 53.8 | 3556 | 1590 | 689 | 2867 | 901 | 137 | 142 | 516 | 231 | |
29.55 | 66.2 | 66.6 | 66.1 | 3678 | 1478 | 685 | 2993 | 793 | 143 | 125 | 537 | 216 | |
24.62 | 77.1 | 76.5 | 77.0 | 3090 | 1408 | 694 | 2396 | 714 | 114 | 113 | 445 | 203 | |
20.52 | 87.7 | 87.5 | 86.6 | 3205 | 1386 | 658 | 2547 | 728 | 122 | 115 | 487 | 211 | |
17.10 | 90.3 | 90.6 | 90.1 | 3040 | 1294 | 640 | 2400 | 654 | 115 | 103 | 475 | 202 | |
14.25 | 90.8 | 90.9 | 91.1 | 3078 | 1277 | 640 | 2438 | 637 | 116 | 101 | 481 | 200 | |
Acceptance Criteria
Acceptance Criterion | Range | Experiment 1 | pass/fail | Experiment 2 | pass/fail | ||||
cell viability solvent controls [%] | >90 | 93.2 | - | 94.5 | pass | 96.7 | - | 97.2 | pass |
number of test dosed with viability >50% CD86 | ≥4 | 4 | pass | 6 | pass | ||||
number of test dosed with viability >50% CD54 | ≥4 | 4 | pass | 6 | pass | ||||
number of test dosed with viability >50% IgG1 | ≥4 | 4 | pass | 6 | pass | ||||
RFI of positive control of CD86 | ≥150 | 273 | pass | 348 | pass | ||||
RFI of positive control of CD54 | ≥200 | 250 | pass | 371 | pass | ||||
RFI of solvent control of CD86 | <150 | 103 | pass | 100 | pass | ||||
RFI of solvent control of CD54 | <200 | 98 | pass | 100 | pass | ||||
MFI ratio IgG1/CD86 for medium control [%] | >105 | 498 | pass | 424 | pass | ||||
MFI ratio IgG1/CD86 for DMSO control [%] | >105 | 500 | pass | 439 | pass | ||||
MFI ratio IgG1/CD54 for medium control [%] | >105 | 211 | pass | 198 | pass | ||||
MFI ratio IgG1/CD54 for DMSO control [%] | >105 | 206 | pass | 202 | pass | ||||
Historical Data
Criterion | mean | SD | N |
cell viability solvent controls [%] | 97.0 | 1.3 | 672 |
number of test doses with viability >50% | - | - | 1786 |
RFI of positive control of CD86 | 401.0 | 146.8 | 112 |
RFI of positive control of CD54 | 576.6 | 312.0 | 112 |
RFI of solvent control of CD86 | 115.0 | 15.1 | 112 |
RFI of solvent control of CD54 | 118.8 | 25.5 | 112 |
MFI ratio IgG1/CD86 for medium control [%] | 202.4 | 50.0 | 112 |
MFI ratio IgG1/CD86 for DMSO control [%] | 221.6 | 58.5 | 112 |
MFI ratio IgG1/CD54 for medium control [%] | 141.0 | 24.7 | 112 |
MFI ratio IgG1/CD54 for DMSO control [%] | 147.7 | 25.6 | 112 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.
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