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EC number: 223-211-1 | CAS number: 3770-97-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Particle size distribution (Granulometry)
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
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Endpoint summary
Administrative data
Description of key information
The skin sensitisation potential was investigated in vitro:
OECD 442C: positive
OECD 442D: negative
OECD 442E: inconclusive
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:
- 2017-11-16 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))
- 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 (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Type of study:
- other: (in chemico) reactivity against synthetic peptides with a thiol or amino group
- 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 64.96%.
- Key result
- Run / experiment:
- other: cysteine run
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 100
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: lysine run
- Parameter:
- other: mean peptide depletion [%]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- 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:
- other: should be considered in the context of integrated approached such as IATA
- Conclusions:
- In this study under the given conditions the test item showed high reactivity towards the cysteine peptide. The test item is considered as “sensitiser”.
The data generated with this method may be not sufficient to conclude on the 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 acetonitrile, based on the results of the pre-experiments.
Based on a molecular weight of 268.5 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.
As the test item undergoes hydrolysis in water, testing might have been performed with reaction products of the test item.
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. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control), as well as for the samples of the test item (excluding the co-elution control of the test item). Samples were centrifuged prior to the HPLC analysis.
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 (excluding 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 precipitations and phase separation were regarded as insignificant.
Co-elution of the test item with the lysine peptide peak was observed. Therefore 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 Cacetonitrile).
The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was > 13.89% (100.00%). Based on the prediction model 2 the test item can be considered as sensitiser.
Although precipitation was observed prior to the HPLC analysis with the cysteine peptide, the obtained positive result can still be used to support the identification of the test chemical as a skin 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 64.96%.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-12-07 to 2018-03-20
- 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 (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 (7.72 (experiment 1); 4.70 (experiment 2)).
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Value:
- 29.27
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 2000 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 3.8
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Value:
- 526.22
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity
- Value:
- 16.99
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 1000 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 6.2
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Value:
- 294.94
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- 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:
- other: should be considered in the context of integrated approached such as IATA
- 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 268.5 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, a max luciferase activity (Imax) induction of 29.27 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 3.8%. The lowest tested concentration with a significant luciferase induction >1.5 (3.92) was found to be 1000 µM. The corresponding cell viability was <70% (34.8%).The calculated EC1.5 was <1000 µM (526.22)
In the second experiment, a max luciferase activity (Imax) induction of 16.99 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 6.2%. The lowest tested concentration with a significant luciferase induction >1.5 (3.37) was found to be 500 µM. The corresponding cell viability was <70% (53.4%).The calculated EC1.5 was < 1000 µM (294.94).
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 luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment one of the technical replicate exceeded the threshold with a fold induction of 12.54. On the one hand, the positive control clearly induces the luciferase induction in the KerationSens™ cell line, showing the capability of the test system to predict sensitisation. On the other hand, the results in this first experiment are clearly negative, indicating no oversensitivity of the test system. The single value of the positive control exceeding the upper threshold was therefore considered to be an outliner and not biological relevant. Furthermore all other controls confirmed the validity of the study
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-07-20 to 2018-10-30
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
- 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 (328% experiment 1; 267% experiment 2, 491% experiment 3, 363% experiment 4, 382% experiment 5) and
200% for CD54 (241% experiment 1; 266% experiment 2, 689% experiment 3, 551% experiment 4 and 297% experiment 5) were clearly exceeded. - Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD86
- Value:
- 84
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 40.06 µg/mL
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 93
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 99.67 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 95
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 40.06 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 102
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 83.06 µg/mL
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 144
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 196.14 µg/mL
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 129
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 338.93 µg/mL
- Key result
- Run / experiment:
- other: 4
- Parameter:
- other: relative fluorescence intensity CD86
- Value:
- 113
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 136.21 µg/mL
- Key result
- Run / experiment:
- other: 4
- Parameter:
- other: relative fluorescence intensity CD54
- Value:
- 148
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration 338.93 µg/mL
- Key result
- Run / experiment:
- other: 5
- Parameter:
- other: relative fluorescence intensity CD86
- Value:
- 281
- Vehicle controls validity:
- not valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 167.45 µg/mL
- Key result
- Run / experiment:
- other: 5
- Parameter:
- other: relative fluorescence intensity CD54
- Value:
- 725
- Vehicle controls validity:
- not valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration 167.45 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. - Conclusions:
- In this study under the given conditions the test item did not upregulate the expression of both cell surface markers which came along with no cytotoxic effects up to the 1.2 x CV75 concentration. Since a negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90% and the highest tested concentration was 1000 µg/mL, this study has to be regarded as inconclusive.
- 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.
Since the CV75 of the dose-finding assay 1 and 2 clearly differed, a third dose-finding assay was performed. Hereby, based on the dose finding assays 2 and 3 a mean CV75 was calculated with 83.06 ± 18.72 µg/mL. Based on the mean CV75, the main experiments 1 and 2 were performed covering the following concentration steps:
99.67, 83.06, 69.22, 57.68, 48.07, 40.06, 33.38, 27.82 µg/mL
Since in the main experiments 1 and 2 no cytotoxic effect and no sensitizing effect was observed, a fourth dose-finding assay was performed in order to verify the CV75. As the CV75 of dose-finding assay 1 and 4 are very comparable, amean CV75 based ondose-finding assay 1 and 4 was calculated with 282.45 ± 6.69 µg/mL. Based on the mean CV75, the main experiments 3 and 4 were performed covering the following concentration steps:
338.93, 282.45, 235.37, 196.14, 163.45, 136.21, 113.51, 94.59 µg/mL
As also for these main experiments 3 and 4 no cytotoxic effect and no sensitizing effect was observed, a fifth dose-finding assay was performed. In this assay, the CV75 ofdose-finding assay 1 and 4 was confirmed.
Therefore, a main experiment 5 using 500 µg/mL as starting concentration was performed covering the following concentration steps:
500, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45, 139.54 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.
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.
No cytotoxic effects were observed for the cells treated with the test item in the main experiments 1, 2, 3 and 4. Relative cell viability at the highest test item concentration was 94.1% (CD86), 94.7% (CD54) and 94.4% (isotype IgG1 control) in the first experiment, 96.1% (CD86), 96.2% (CD54) and 95.6% (isotype IgG1 control) in the second experiment, 95.6% (CD86), 95.6% (CD54) and 95.5% (isotype IgG1 control) in the third experiment and 94.1% (CD86), 94.2% (CD54) and 93.8% (isotype IgG1 control) in the fourth experiment. Cytotoxic effects were observed for the cells treated with the test item in main experiment 5. Relative cell viability at the highest test item concentration was reduced to 22.8% (CD86), 20.5% (CD54) and 20.2% (isotype IgG1 control) in the fifth experiment.
In the experiments 1, 2, 3 and 4 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. However, since a negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90% and the highest tested concentration was 1000 µg/mL, the experiments1, 2, 3 and 4 have to be regarded as inconclusive.
In experiment 5 the expression of the cell surface marker CD86 was upregulated above the threshold of 150% to a maximum of 281% at a concentration of 167.45 µg/mL.The expression of cell surface marker CD54 was upregulated above the threshold of 200% to a maximum of 725% at a concentration of 167.45 µg/mL. However, as only two concentrations showed a viability >50%, this experiment 5 was not valid.
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 |
4311.3857 |
0.5340 |
STD2 |
2255.1311 |
0.2670 |
2143.2170 |
0.2670 |
STD3 |
1081.2599 |
0.1335 |
1043.3549 |
0.1335 |
STD4 |
538.3588 |
0.0667 |
519.3511 |
0.0667 |
STD5 |
264.6839 |
0.0334 |
259.5146 |
0.0334 |
STD6 |
131.3584 |
0.0167 |
130.4601 |
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 |
0.0000 |
0.0023 |
100.00 |
100.00 |
0.00 |
0.00 |
0.0000 |
0.0023 |
100.00 |
||||
0.0000 |
0.0023 |
100.00 |
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 |
1589.2585 |
0.1982 |
59.95 |
58.82 |
0.99 |
1.68 |
1662.4030 |
0.2073 |
58.11 |
||||
1650.5492 |
0.2058 |
58.41 |
||||
Test Item* |
- |
- |
- |
- |
- |
n/a |
- |
- |
- |
||||
- |
- |
- |
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 |
- |
n/a |
n/a |
100.00 |
High Reactivity |
sensitiser |
Positive Control |
64.96 |
High Reactivity |
sensitiser |
71.10 |
Moderate Reactivity |
sensitiser |
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 |
100.4 |
87.0 |
93.7 |
9.5 |
8.00 |
98.5 |
66.4 |
82.5 |
22.7 |
|
16.00 |
97.0 |
76.3 |
86.6 |
14.6 |
|
32.00 |
95.4 |
59.3 |
77.4 |
25.5 |
|
64.00 |
95.8 |
48.8 |
72.3 |
33.2 |
|
Test Item |
0.98 |
94.1 |
107.1 |
100.6 |
9.2 |
1.95 |
94.0 |
101.5 |
97.8 |
5.3 |
|
3.91 |
90.9 |
101.1 |
96.0 |
7.2 |
|
7.81 |
89.8 |
100.8 |
95.3 |
7.8 |
|
15.63 |
88.4 |
100.0 |
94.2 |
8.3 |
|
31.25 |
90.4 |
93.8 |
92.1 |
2.4 |
|
62.50 |
79.7 |
85.6 |
82.7 |
4.2 |
|
125.00 |
53.5 |
63.2 |
58.3 |
6.9 |
|
250.00 |
40.8 |
47.8 |
44.3 |
5.0 |
|
500.00 |
37.2 |
53.4 |
45.3 |
11.5 |
|
1000.00 |
34.8 |
6.2 |
20.5 |
20.2 |
|
2000.00 |
3.8 |
2.9 |
3.4 |
0.7 |
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 |
1.28 |
1.23 |
1.23 |
1.25 |
0.03 |
|
8.00 |
1.44 |
1.35 |
1.29 |
1.36 |
0.07 |
|
|
16.00 |
1.82 |
1.66 |
1.50 |
1.66 |
0.16 |
* |
|
32.00 |
2.52 |
2.75 |
1.68 |
2.32 |
0.57 |
* |
|
64.00 |
7.62 |
(12.54#) |
7.82 |
7.72 |
0.10 |
* |
|
Test Item |
0.98 |
1.03 |
1.04 |
1.07 |
1.05 |
0.02 |
|
1.95 |
0.99 |
1.01 |
0.92 |
0.97 |
0.05 |
|
|
3.91 |
1.08 |
1.06 |
0.95 |
1.03 |
0.07 |
|
|
7.81 |
1.16 |
1.27 |
1.14 |
1.19 |
0.07 |
|
|
15.63 |
1.00 |
1.08 |
1.01 |
1.03 |
0.04 |
|
|
31.25 |
1.03 |
1.07 |
0.93 |
1.01 |
0.07 |
|
|
62.50 |
1.04 |
1.16 |
0.95 |
1.05 |
0.10 |
|
|
125.00 |
1.09 |
0.95 |
0.88 |
0.97 |
0.11 |
|
|
250.00 |
1.03 |
1.15 |
0.80 |
0.99 |
0.18 |
|
|
500.00 |
1.30 |
1.51 |
1.29 |
1.37 |
0.13 |
|
|
1000.00 |
3.56 |
5.19 |
3.02 |
3.92 |
1.13 |
* |
|
2000.00 |
38.70 |
33.84 |
15.27 |
29.27 |
12.37 |
* |
* = significant induction according to Student’s t-test, p < 0.05
#= outlier according to Grubbs, Nalimov and Dixon. Excluded from evaluation.
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 |
1.16 |
1.18 |
1.10 |
1.15 |
0.04 |
|
8.00 |
1.16 |
1.10 |
1.09 |
1.12 |
0.04 |
|
|
16.00 |
1.65 |
1.69 |
1.49 |
1.61 |
0.11 |
* |
|
32.00 |
2.23 |
1.88 |
1.93 |
2.01 |
0.19 |
* |
|
64.00 |
4.84 |
4.51 |
4.75 |
4.70 |
0.17 |
* |
|
Test Item |
0.98 |
1.17 |
1.20 |
1.00 |
1.13 |
0.11 |
|
1.95 |
0.97 |
1.03 |
0.88 |
0.96 |
0.08 |
|
|
3.91 |
0.97 |
0.91 |
0.99 |
0.96 |
0.04 |
|
|
7.81 |
0.87 |
0.92 |
0.82 |
0.87 |
0.05 |
|
|
15.63 |
0.88 |
0.98 |
1.07 |
0.98 |
0.09 |
|
|
31.25 |
0.87 |
0.81 |
0.81 |
0.83 |
0.04 |
|
|
62.50 |
0.93 |
0.95 |
0.78 |
0.89 |
0.09 |
|
|
125.00 |
1.09 |
1.06 |
0.85 |
1.00 |
0.13 |
|
|
250.00 |
1.27 |
1.04 |
0.96 |
1.09 |
0.16 |
|
|
500.00 |
6.05 |
2.42 |
1.64 |
3.37 |
2.35 |
|
|
1000.00 |
9.30 |
29.65 |
12.01 |
16.99 |
11.05 |
* |
|
2000.00 |
0.01 |
0.01 |
1.52 |
0.51 |
0.87 |
|
* = 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 |
1.25 |
1.15 |
1.20 |
0.07 |
|
8.00 |
1.36 |
1.12 |
1.24 |
0.17 |
|
|
16.00 |
1.66 |
1.61 |
1.63 |
0.04 |
* |
|
32.00 |
2.32 |
2.01 |
2.16 |
0.22 |
* |
|
64.00 |
9.33 |
4.70 |
7.01 |
3.27 |
|
|
Test Item |
0.98 |
1.05 |
1.13 |
1.09 |
0.06 |
|
1.95 |
0.97 |
0.96 |
0.97 |
0.01 |
|
|
3.91 |
1.03 |
0.96 |
0.99 |
0.05 |
|
|
7.81 |
1.19 |
0.87 |
1.03 |
0.22 |
|
|
15.63 |
1.03 |
0.98 |
1.00 |
0.04 |
|
|
31.25 |
1.01 |
0.83 |
0.92 |
0.13 |
|
|
62.50 |
1.05 |
0.89 |
0.97 |
0.12 |
|
|
125.00 |
0.97 |
1.00 |
0.99 |
0.02 |
|
|
250.00 |
0.99 |
1.09 |
1.04 |
0.07 |
|
|
500.00 |
1.37 |
3.37 |
2.37 |
1.42 |
|
|
1000.00 |
3.92 |
16.99 |
10.45 |
9.24 |
|
|
2000.00 |
29.27 |
0.51 |
14.89 |
20.33 |
|
* = significant induction according to Student’s t-test, p < 0.05
Additional Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Mean |
SD |
EC1.5[µM] |
526.22 |
294.94 |
410.58 |
163.54 |
Imax |
29.27 |
16.99 |
23.13 |
8.69 |
IC30[µM] |
85.61 |
106.04 |
95.82 |
14.45 |
IC50[µM] |
159.32 |
231.00 |
195.16 |
50.69 |
Acceptance Criteria
Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
CV Solvent Control |
< 20% |
13.1 |
pass |
9.6 |
pass |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
3.0 |
pass |
3.0 |
pass |
EC1.5 PC |
7 < x < 34 µM |
11.72 |
pass |
14.23 |
pass |
Induction PC at 64 µM |
2.00 < x < 8.00 |
7.72* |
pass |
4.70 |
pass |
* = Mean out of replicate 1 and 3. 2 replicate is an outlier according to Grubbs, Nalimov and Dixon.
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 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 Cell Batch Activation Test (Batch 22)
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 |
85.3 |
329 |
>150 |
85.1 |
434 |
>200 |
yes |
pass |
NiSO4old |
100 µg/mL |
88.6 |
294 |
>150 |
89.4 |
447 |
>200 |
yes |
pass |
NiSO4new |
100 µg/mL |
88.7 |
298 |
>150 |
88.7 |
456 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
96.7 |
84 |
≤150 |
96.6 |
107 |
≤200 |
no |
pass |
Results of the Cell Batch Activation Test (Batch 01)
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 |
87.5 |
373 |
>150 |
88.1 |
358 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
83.5 |
295 |
>150 |
82.1 |
603 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
95.7 |
81 |
≤150 |
95.8 |
100 |
≤200 |
no |
pass |
Results of the Cell Batch Activation Test (Batch 02)
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 |
83.4 |
364 |
>150 |
82.3 |
419 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
79.1 |
351 |
>150 |
77.9 |
688 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
96.2 |
77 |
≤150 |
95.9 |
105 |
≤200 |
no |
pass |
Results of the Dose Finding Assays
Sample |
Cell Viability |
||||||
Concentration applied [µg/mL] |
Assay 1 |
Assay 2 |
Assay 3 |
Assay 4 |
Assay 5 |
||
Medium Control |
-- |
-- |
94.60 |
93.90 |
95.60 |
95.50 |
96.50 |
Solvent Control |
DMSO |
-- |
94.30 |
93.90 |
95.20 |
96.20 |
93.90 |
F6-Cl |
C8 |
7.81 |
94.20 |
92.10 |
96.30 |
95.70 |
94.60 |
C7 |
15.63 |
94.50 |
92.60 |
95.70 |
96.00 |
94.30 |
|
C6 |
31.25 |
94.40 |
93.30 |
95.10 |
96.50 |
95.30 |
|
C5 |
62.50 |
94.10 |
91.40 |
87.90 |
96.00 |
94.40 |
|
C4 |
125.00 |
93.70 |
65.10 |
7.20 |
95.20 |
93.50 |
|
C3 |
250.00 |
91.10 |
16.10 |
4.80 |
85.80 |
68.30 |
|
C2 |
500.00 |
10.60 |
19.50 |
26.20 |
14.60 |
23.20 |
|
C1 |
1000.00 |
11.20 |
26.40 |
10.20 |
9.00 |
14.40 |
|
Calculated CV75 [µg/mL] |
-- |
287.17 |
96.29 |
69.82 |
277.72 |
207.92 |
CD54 and CD86 Expression Experiment 1
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Fluorescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
96.8 |
95.5 |
95.7 |
2078 |
1010 |
583 |
1495 |
427 |
98 |
87 |
356 |
173 |
Solvent Control |
0.20% |
96.2 |
96.0 |
96.1 |
2087 |
1057 |
565 |
1522 |
492 |
100 |
100 |
369 |
187 |
DNCB |
4.00 |
86.3 |
86.9 |
86.0 |
5604 |
1790 |
606 |
4998 |
1184 |
328 |
241 |
925 |
295 |
F6-Cl |
99.67 |
94.1 |
94.7 |
94.4 |
1900 |
1089 |
633 |
1267 |
456 |
83 |
93 |
300 |
172 |
83.06 |
95.6 |
95.1 |
95.8 |
1780 |
1013 |
672 |
1108 |
341 |
73 |
69 |
265 |
151 |
|
69.22 |
95.6 |
95.7 |
95.2 |
1735 |
1040 |
631 |
1104 |
409 |
73 |
83 |
275 |
165 |
|
57.68 |
95.1 |
95.2 |
95.1 |
1802 |
1011 |
606 |
1196 |
405 |
79 |
82 |
297 |
167 |
|
48.07 |
96.7 |
96.2 |
95.7 |
1697 |
1014 |
611 |
1086 |
403 |
71 |
82 |
278 |
166 |
|
40.06 |
95.5 |
95.4 |
95.5 |
1882 |
1005 |
611 |
1271 |
394 |
84 |
80 |
308 |
164 |
|
33.38 |
95.2 |
95.2 |
95.3 |
1856 |
977 |
626 |
1230 |
351 |
81 |
71 |
296 |
156 |
|
27.82 |
95.4 |
95.3 |
95.3 |
1749 |
983 |
577 |
1172 |
406 |
77 |
83 |
303 |
170 |
CD54 and CD86 Expression Experiment 2
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Fluorescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
97.4 |
97.0 |
97.2 |
1920 |
864 |
516 |
1404 |
348 |
79 |
96 |
372 |
167 |
Solvent Control |
0.20% |
96.8 |
96.8 |
96.3 |
2286 |
878 |
514 |
1772 |
364 |
100 |
100 |
445 |
171 |
DNCB |
4.0 |
87.8 |
86.9 |
87.8 |
5309 |
1539 |
572 |
4737 |
967 |
267 |
266 |
928 |
269 |
F6-Cl |
99.67 |
96.1 |
96.2 |
95.6 |
2001 |
917 |
571 |
1430 |
346 |
81 |
95 |
350 |
161 |
83.06 |
95.9 |
95.8 |
96.2 |
1996 |
934 |
564 |
1432 |
370 |
81 |
102 |
354 |
166 |
|
69.22 |
96.7 |
96.7 |
95.9 |
1896 |
928 |
574 |
1322 |
354 |
75 |
97 |
330 |
162 |
|
57.68 |
96.3 |
95.7 |
95.8 |
2087 |
920 |
566 |
1521 |
354 |
86 |
97 |
369 |
163 |
|
48.07 |
96.4 |
96.1 |
96.0 |
2006 |
918 |
555 |
1451 |
363 |
82 |
100 |
361 |
165 |
|
40.06 |
95.7 |
96.2 |
95.6 |
2223 |
899 |
537 |
1686 |
362 |
95 |
99 |
414 |
167 |
|
33.38 |
96.2 |
96.1 |
95.7 |
1819 |
849 |
514 |
1305 |
335 |
74 |
92 |
354 |
165 |
|
27.82 |
95.7 |
95.7 |
95.8 |
1794 |
816 |
501 |
1293 |
315 |
73 |
87 |
358 |
163 |
CD54 and CD86 Expression Experiment 3
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Fluorescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
96.4 |
96.2 |
96.3 |
1374 |
832 |
502 |
872 |
330 |
91 |
97 |
274 |
166 |
Solvent Control |
0.20% |
96.1 |
95.5 |
96.1 |
1452 |
832 |
493 |
959 |
339 |
100 |
100 |
295 |
169 |
DNCB |
4.00 |
82.5 |
82.9 |
82.2 |
5346 |
2974 |
639 |
4707 |
2335 |
491 |
689 |
837 |
465 |
F6-Cl |
338.93 |
95.6 |
95.6 |
95.5 |
1551 |
1113 |
676 |
875 |
437 |
91 |
129 |
229 |
165 |
282.44 |
96.3 |
95.7 |
95.9 |
1619 |
1110 |
688 |
931 |
422 |
97 |
124 |
235 |
161 |
|
235.37 |
96.3 |
96.0 |
95.9 |
1609 |
1046 |
629 |
980 |
417 |
102 |
123 |
256 |
166 |
|
196.14 |
96.5 |
96.2 |
95.9 |
2012 |
1058 |
627 |
1385 |
431 |
144 |
127 |
321 |
169 |
|
163.45 |
96.5 |
96.0 |
96.1 |
1392 |
977 |
613 |
779 |
364 |
81 |
107 |
227 |
159 |
|
136.21 |
96.5 |
96.6 |
96.3 |
1606 |
966 |
603 |
1003 |
363 |
105 |
107 |
266 |
160 |
|
113.51 |
96.4 |
96.4 |
96.2 |
1791 |
991 |
596 |
1195 |
395 |
125 |
117 |
301 |
166 |
|
94.59 |
96.6 |
95.1 |
95.9 |
1601 |
964 |
597 |
1004 |
367 |
105 |
108 |
268 |
161 |
CD54 and CD86 Expression Experiment 4
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Fluorescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
95.7 |
96.7 |
96.3 |
1301 |
751 |
537 |
764 |
214 |
81 |
83 |
242 |
140 |
Solvent Control |
0.20% |
96.2 |
96.6 |
96.5 |
1450 |
767 |
508 |
942 |
259 |
100 |
100 |
285 |
151 |
DNCB |
4.0 |
87.5 |
87.7 |
87.3 |
4043 |
2054 |
628 |
3415 |
1426 |
363 |
551 |
644 |
327 |
F6-Cl |
338.93 |
94.1 |
94.2 |
93.8 |
1760 |
1105 |
721 |
1039 |
384 |
110 |
148 |
244 |
153 |
282.44 |
94.6 |
95.2 |
94.6 |
1709 |
1054 |
698 |
1011 |
356 |
107 |
137 |
245 |
151 |
|
235.37 |
94.7 |
95.1 |
95.4 |
1706 |
1020 |
682 |
1024 |
338 |
109 |
131 |
250 |
150 |
|
196.14 |
96.2 |
96.3 |
96.6 |
1575 |
1035 |
671 |
904 |
364 |
96 |
141 |
235 |
154 |
|
163.45 |
95.3 |
94.9 |
95.8 |
1672 |
946 |
629 |
1043 |
317 |
111 |
122 |
266 |
150 |
|
136.21 |
96.0 |
96.0 |
94.9 |
1702 |
986 |
641 |
1061 |
345 |
113 |
133 |
266 |
154 |
|
113.51 |
95.8 |
95.8 |
96.1 |
1550 |
895 |
599 |
951 |
296 |
101 |
114 |
259 |
149 |
|
94.59 |
96.4 |
96.4 |
96.4 |
1411 |
673 |
554 |
857 |
119 |
91 |
46 |
255 |
121 |
CD54 and CD86 Expression Experiment 5
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Fluorescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
96.7 |
96.1 |
95.2 |
1773 |
992 |
548 |
1225 |
444 |
100 |
93 |
324 |
181 |
Solvent Control |
0.20% |
95.7 |
95.6 |
95.0 |
1756 |
1007 |
531 |
1225 |
476 |
100 |
100 |
331 |
190 |
DNCB |
4.0 |
85.2 |
85.2 |
84.5 |
5286 |
2023 |
611 |
4675 |
1412 |
382 |
297 |
865 |
331 |
F6-Cl |
500.0 |
22.8 |
20.5 |
20.2 |
4933 |
4133 |
3818 |
1115 |
315 |
91 |
66 |
129 |
108 |
416.67 |
22.2 |
20.7 |
20.5 |
5153 |
4198 |
3927 |
1226 |
271 |
100 |
57 |
131 |
107 |
|
347.22 |
24.2 |
22.7 |
22.5 |
4713 |
2707 |
1958 |
2755 |
749 |
225 |
157 |
241 |
138 |
|
289.35 |
19.4 |
18.1 |
17.5 |
5687 |
4435 |
4060 |
1627 |
375 |
133 |
79 |
140 |
109 |
|
241.13 |
18.6 |
18.2 |
17.3 |
5745 |
4400 |
3971 |
1774 |
429 |
145 |
90 |
145 |
111 |
|
200.94 |
21.6 |
20.3 |
18.6 |
5466 |
4055 |
3606 |
1860 |
449 |
152 |
94 |
152 |
112 |
|
167.45 |
54.1 |
52.8 |
52.3 |
4473 |
4480 |
1031 |
3442 |
3449 |
281 |
725 |
434 |
435 |
|
139.54 |
85.1 |
82.1 |
84.1 |
2740 |
1655 |
673 |
2067 |
982 |
169 |
206 |
407 |
246 |
Acceptance Criteria experiment 1 and 2
Acceptance Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
||||
cell viability solvent controls [%] |
>90 |
95.5 |
- |
96.8 |
pass |
96.3 |
- |
97.4 |
pass |
number of test dosed with viability >50% CD86 |
≥4 |
8 |
pass |
8 |
pass |
||||
number of test dosed with viability >50% CD54 |
≥4 |
8 |
pass |
8 |
pass |
||||
number of test dosed with viability >50% IgG1 |
≥4 |
8 |
pass |
8 |
pass |
||||
RFI of positive control of CD86 |
≥150 |
328 |
pass |
267 |
pass |
||||
RFI of positive control of CD54 |
≥200 |
241 |
pass |
266 |
pass |
||||
RFI of solvent control of CD86 |
<150 |
102 |
pass |
126 |
pass |
||||
RFI of solvent control of CD54 |
<200 |
115 |
pass |
105 |
pass |
||||
MFI ratio CD86/IgG1 for medium control [%] |
>105 |
356 |
pass |
372 |
pass |
||||
MFI ratio CD86/IgG1 for DMSO control [%] |
>105 |
369 |
pass |
445 |
pass |
||||
MFI ratio CD54/IgG1for medium control [%] |
>105 |
173 |
pass |
167 |
pass |
||||
MFI ratio CD54/IgG1for DMSO control [%] |
>105 |
187 |
pass |
171 |
pass |
||||
Acceptance Criteria experiment 3, 4 and 5
Acceptance Criterion |
Range |
Experiment 3 |
pass/fail |
Experiment 4 |
pass/fail |
Experiment 5 |
pass/fail |
||||||
cell viability solvent controls [%] |
>90 |
95.5 |
- |
96.4 |
pass |
95.7 |
- |
96.7 |
pass |
95.0 |
- |
96.7 |
pass |
number of test dosed with viability >50% CD86 |
≥4 |
8 |
pass |
8 |
pass |
2 |
fail |
||||||
number of test dosed with viability >50% CD54 |
≥4 |
8 |
pass |
8 |
pass |
2 |
fail |
||||||
number of test dosed with viability >50% IgG1 |
≥4 |
8 |
pass |
8 |
pass |
2 |
fail |
||||||
RFI of positive control of CD86 |
≥150 |
491 |
pass |
363 |
pass |
382 |
pass |
||||||
RFI of positive control of CD54 |
≥200 |
689 |
pass |
551 |
pass |
297 |
pass |
||||||
RFI of solvent control of CD86 |
<150 |
110 |
pass |
123 |
pass |
100 |
pass |
||||||
RFI of solvent control of CD54 |
<200 |
103 |
pass |
121 |
pass |
107 |
pass |
||||||
MFI ratio CD86/IgG1 for medium control [%] |
>105 |
274 |
pass |
242 |
pass |
324 |
pass |
||||||
MFI ratio CD86/IgG1 for DMSO control [%] |
>105 |
295 |
pass |
285 |
pass |
331 |
pass |
||||||
MFI ratio CD54/IgG1for medium control [%] |
>105 |
166 |
pass |
140 |
pass |
181 |
pass |
||||||
MFI ratio CD54/IgG1for DMSO control [%] |
>105 |
169 |
pass |
151 |
pass |
190 |
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:
- adverse effect observed (sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the provided data, a clear positive response in the DPRA assay was observed. No conclusion can be made regarding the classification for skin sensitization based on the results of the OECD 442E assay. The OECD 442D assay was negative. The compound is highly reactive when exposed to light or in aqueous environment. Therefore, binding to skin proteins can be assumed leading to hapten formation as initial step of skin sensitisation. Taken together the high reactivity of the compound and the results from the DPRA assay suggest that a classification for skin sensitisation class 1 is fully justified.
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