Registration Dossier
Registration Dossier
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EC number: 836-530-1 | CAS number: 1296134-40-1
Endpoint summary
Administrative data
Description of key information
Skin sensitisation is considered as an allergic response following skin contact. The sensitisation potential can be assessed by numerous assays including in vivo studies and in vitro
experiments.
The in vitro assays performed show that due to the physicochemical properties of the compound, namely logP and limited solubility, the in vitro approach is technically challenging. Two of the three assays performed are inconclusive thus a conclusion based on in vitro data is not possible.
However, QSAR analysis of the chemical structure provides additional information. This analysis resulted in two structural alerts for skin sensitisation. The subsequent EC3 prediction based on information of close structural analogues reveals a moderate potential for skin sensitisation.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in chemico
- Study period:
- 09/2019
- Data waiving:
- study technically not feasible
- Justification for data waiving:
- other:
- Justification for type of information:
- The test item solubility was tested in acetonitrile, water, isopropanol, methanol, ethanol, 1,4-butandiol, N,Ndimethylformamide and tert-butanol. The solubility of the test item was tested at 100 mM. The test item was not soluble in any of all suitable solvents. Therefore, the study cannot be performed.
- Interpretation of results:
- study cannot be used for classification
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-09-13 to 2018-10-01
- 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 (6.02 (experiment 1) and 4.40 (experiment 2)).
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Value:
- 1.22
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 0.98 µM
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 42.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- 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: luciferase activity
- Value:
- 1.09
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 0.98 µM
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 65.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- 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
- 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: The result should be considered in the context of integrated approach 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 THF. Based on a molecular weight of 1628 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.
Microscopically, precipitates of the test item were observed after the incubation in the medium for the concentrations from 7.81 µM to 2000 µM. In the first main experiment cytotoxic effects were observed but no dose response was obtained. In the second experiment no cytotoxic effects were observed. Therefore, it was assumed that the cytotoxic effects in the first experiment were caused by the precipitations. Furthermore, no cytotoxic effects were observed microscopically. 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:
- 2019-01-23 to 2019-02-28
- 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 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 all three experiments. The threshold of 150% for CD86 (301% experiment 1; 242% experiment 2; 288% experiment 3) and 200% for CD54 (256% experiment 1; 292% experiment 2; 396% experiment 3) were clearly exceeded.
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 148
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 482.25 µg/mL
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 115
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 482.25 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 151
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 279.08 µg/mL
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 110
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 279.08 µg/mL
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 110
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 1000 µg/mL
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 131
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Concentration: 401.88 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:
- other: The results should be considered in the context of integrated approach such as IATA
- 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. Furthermore, due to the high Log KOW the test item has to be considered as inconclusive. 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 item was dissolved in THF. 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.
Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:
1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL
In all experiments precipitation 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. Relative cell viability at the highest test item concentration was 96.9% (CD86), 96.9% (CD54) and 97.0% (isotype IgG1 control) in the first experiment, 92.2% (CD86), 92.1% (CD54) and 92.0% (isotype IgG1 control) in the second experiment and 96.6% (CD86), 96.3% (CD54) and 96.1% (isotype IgG1 control) in the third experiment.
The expression of the cell surface marker CD86 was upregulated to 151% only in the second experiment. The upregulation above the threshold of 150% was observed at a concentration of 279.08 µg/mL. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.
Since the cell surface marker CD86 exceeded the threshold only in one of the three independent experiments, the test item could be considered to be a non-sensitiser. Due to the strong observed precipitation in all experiments an interaction between the test item and the cells cannot be guaranteed and a negative result must be considered carefully. Furthermore, due to the high Log Kow the test item has to be considered as inconclusive.
- Endpoint:
- skin sensitisation, other
- Remarks:
- in silico
- Type of information:
- (Q)SAR
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Justification for type of information:
- Please refer to the QMRF and QPRF files provided under the section attached justification.
- Qualifier:
- according to guideline
- Guideline:
- other: ECHA Guidance on IR/CSA R.6 QSARs and grouping of chemicals
- Principles of method if other than guideline:
- Estimates the skin sensitising properties of chemicals using structural alert relationships.
- GLP compliance:
- no
- Specific details on test material used for the study:
- see QPRF
- Key result
- Parameter:
- other: Structural Alert for skin sensitisaton
- Value:
- 2
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- Two alerts for skin sensitisation have been identified. See QPRF for details.
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- Using Derek and Meteor Nexus , the skin sensitising potential assessment resulted in two alerts for skin sensitisation. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate. Due to the prediction result, the compound should be regarded as moderate skin sensitiser.
- Endpoint:
- skin sensitisation, other
- Type of information:
- other: Weight of Evidence Justification
- Adequacy of study:
- other information
- Study period:
- 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Key result
- Parameter:
- other:
- Value:
- 1
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- The structural alerts identified in the QSAR analysis and the moderate EC3 potential for skin sensitisation estimated from the data of structural analogues provide sufficient evi-dence for a classification in class 1B for skin sensitisation for the test item.
Referenceopen allclose all
Results of the Cytotoxicity Measurement
|
Concentration [µM] |
Cell Viability [%] |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
||
Positive Control |
4.00 |
93.7 |
94.2 |
93.9 |
0.4 |
8.00 |
97.8 |
95.2 |
96.5 |
1.8 |
|
16.00 |
102.6 |
98.8 |
100.7 |
2.7 |
|
32.00 |
103.8 |
96.8 |
100.3 |
4.9 |
|
64.00 |
103.8 |
93.6 |
98.7 |
7.2 |
|
Test Item |
0.98 |
42.9 |
65.5 |
54.2 |
16.0 |
1.95 |
89.8 |
80.2 |
85.0 |
6.8 |
|
3.91 |
81.2 |
93.7 |
87.4 |
8.8 |
|
7.81 |
76.3 |
88.6 |
82.5 |
8.7 |
|
15.63 |
79.0 |
92.4 |
85.7 |
9.4 |
|
31.25 |
60.5 |
90.2 |
75.4 |
21.0 |
|
62.50 |
70.8 |
90.6 |
80.7 |
14.0 |
|
125.00 |
78.3 |
84.6 |
81.5 |
4.5 |
|
250.00 |
56.7 |
91.3 |
74.0 |
24.5 |
|
500.00 |
80.4 |
91.4 |
85.9 |
7.8 |
|
1000.00 |
85.3 |
91.2 |
88.3 |
4.2 |
|
2000.00 |
78.5 |
91.5 |
85.0 |
9.2 |
|
Induction of Luciferase Activity Experiment 1
Experiment 1 |
Concentration [µM] |
Fold Induction |
Significance |
||||
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|||
Positive Control |
4.00 |
1.03 |
1.07 |
1.09 |
1.07 |
0.03 |
|
8.00 |
1.29 |
1.31 |
1.39 |
1.33 |
0.06 |
|
|
16.00 |
1.54 |
1.76 |
1.65 |
1.65 |
0.11 |
* |
|
32.00 |
2.51 |
2.09 |
2.48 |
2.36 |
0.23 |
* |
|
64.00 |
6.93 |
4.60 |
6.53 |
6.02 |
1.25 |
* |
|
Test Item |
0.98 |
1.17 |
1.16 |
1.32 |
1.22 |
0.09 |
|
1.95 |
0.91 |
1.43 |
1.10 |
1.14 |
0.27 |
|
|
3.91 |
0.84 |
1.04 |
1.23 |
1.04 |
0.19 |
|
|
7.81 |
0.79 |
1.03 |
1.17 |
1.00 |
0.19 |
|
|
15.63 |
0.78 |
1.10 |
1.27 |
1.05 |
0.25 |
|
|
31.25 |
0.75 |
0.99 |
1.07 |
0.94 |
0.17 |
|
|
62.50 |
0.90 |
1.06 |
1.04 |
1.00 |
0.09 |
|
|
125.00 |
0.90 |
0.92 |
0.94 |
0.92 |
0.02 |
|
|
250.00 |
1.36 |
1.34 |
0.94 |
1.21 |
0.24 |
|
|
500.00 |
0.96 |
1.00 |
0.90 |
0.96 |
0.05 |
|
|
1000.00 |
1.02 |
1.04 |
0.99 |
1.02 |
0.03 |
|
|
2000.00 |
0.99 |
1.06 |
0.90 |
0.98 |
0.08 |
|
|
* = significant induction according to Student’s t-test, p<0.05
Induction of Luciferase Activity Experiment 2
Experiment 2 |
Concentration [µM] |
Fold Induction |
Significance |
||||
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|||
Positive Control |
4.00 |
1.12 |
1.16 |
1.23 |
1.17 |
0.05 |
|
8.00 |
1.29 |
1.17 |
1.14 |
1.20 |
0.08 |
|
|
16.00 |
1.41 |
1.53 |
1.50 |
1.48 |
0.06 |
|
|
32.00 |
1.80 |
2.02 |
2.26 |
2.03 |
0.23 |
* |
|
64.00 |
4.08 |
3.90 |
5.21 |
4.40 |
0.71 |
* |
|
Test Item |
0.98 |
0.73 |
1.31 |
1.23 |
1.09 |
0.32 |
|
1.95 |
0.88 |
1.12 |
0.96 |
0.99 |
0.12 |
|
|
3.91 |
0.81 |
1.10 |
0.95 |
0.96 |
0.15 |
|
|
7.81 |
0.96 |
1.24 |
0.97 |
1.06 |
0.16 |
|
|
15.63 |
0.85 |
1.02 |
1.00 |
0.95 |
0.09 |
|
|
31.25 |
0.82 |
0.94 |
1.12 |
0.96 |
0.15 |
|
|
62.50 |
0.68 |
1.09 |
1.19 |
0.99 |
0.27 |
|
|
125.00 |
0.91 |
0.99 |
1.00 |
0.97 |
0.05 |
|
|
250.00 |
0.77 |
1.02 |
1.16 |
0.98 |
0.20 |
|
|
500.00 |
0.78 |
0.92 |
1.02 |
0.91 |
0.12 |
|
|
1000.00 |
0.84 |
1.17 |
1.16 |
1.06 |
0.18 |
|
|
2000.00 |
0.93 |
1.09 |
1.23 |
1.08 |
0.15 |
|
|
* = significant induction according to Student’s t-test, p<0.05
Induction of Luciferase Activity – Overall Induction
|
Concentration [µM] |
Fold Induction |
|||
Experiment 1 |
Experiment 2 |
Mean |
SD |
||
Positive Control |
4.00 |
1.07 |
1.17 |
1.12 |
0.07 |
8.00 |
1.33 |
1.20 |
1.26 |
0.09 |
|
16.00 |
1.65 |
1.48 |
1.56 |
0.12 |
|
32.00 |
2.36 |
2.03 |
2.19 |
0.23 |
|
64.00 |
6.02 |
4.40 |
5.21 |
1.15 |
|
Test Item |
0.98 |
1.22 |
1.09 |
1.15 |
0.09 |
1.95 |
1.14 |
0.99 |
1.07 |
0.11 |
|
3.91 |
1.04 |
0.96 |
1.00 |
0.06 |
|
7.81 |
1.00 |
1.06 |
1.03 |
0.04 |
|
15.63 |
1.05 |
0.95 |
1.00 |
0.07 |
|
31.25 |
0.94 |
0.96 |
0.95 |
0.02 |
|
62.50 |
1.00 |
0.99 |
0.99 |
0.01 |
|
125.00 |
0.92 |
0.97 |
0.94 |
0.03 |
|
250.00 |
1.21 |
0.98 |
1.10 |
0.16 |
|
500.00 |
0.96 |
0.91 |
0.93 |
0.03 |
|
1000.00 |
1.02 |
1.06 |
1.04 |
0.03 |
|
2000.00 |
0.98 |
1.08 |
1.03 |
0.07 |
|
* = significant induction according to Student’s t-test, p<0.05
Additional Parameters
Parameter |
Experiment 1 |
Experiment 2 |
Mean |
SD |
EC1.5[µM] |
n.a. |
n.a. |
n.a. |
n.a. |
Imax |
1.22 |
1.09 |
1.15 |
0.09 |
IC30[µM] |
n.a. |
n.a. |
n.a. |
n.a. |
IC50[µM] |
n.a. |
n.a. |
n.a. |
n.a. |
n.a.: not applicable
Acceptance Criteria
Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
CV Solvent Control PC (1% DMSO) |
< 20% |
4.0 |
pass |
12.4 |
pass |
CV Solvent Control TI (1% THF) |
<20% |
9.0 |
pass |
12.6 |
pass |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
3.0 |
pass |
2.0 |
pass |
EC1.5 PC |
7 < x < 34 µM |
12.28 |
pass |
16.57 |
pass |
Induction PC at 64 µM |
2.00 < x < 8.00 |
6.02 |
pass |
4.40 |
pass |
Historical Data
Acceptance Criterion |
Range |
Mean |
SD |
N |
CV Solvent Control |
< 20% |
11.6 |
3.5 |
96 |
No. of positive control concentration steps with significant luciferase activity induction >1.5 |
≥ 1 |
2.4 |
0.6 |
96 |
EC1.5 PC |
7 < x < 34 µM |
18.5 |
6.0 |
96 |
Induction PC at 64 µM |
2.00 < x < 8.00 |
3.8 |
1.5 |
96 |
Results of the Cell Batch Activation Test (Batch 03)
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 |
85.4 |
306 |
>150 |
84.7 |
220 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
81.8 |
251 |
>150 |
80.9 |
363 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
94.0 |
93 |
≤150 |
93.9 |
93 |
≤200 |
no |
pass |
Results of the Cell Batch Activation Test (Batch 04)
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 |
84.5 |
342 |
>150 |
85.8 |
334 |
>200 |
yes |
pass |
NiSO4 |
100 µg/mL |
81.7 |
280 |
>150 |
81.7 |
578 |
>200 |
yes |
pass |
LA |
1000 µg/mL |
95.3 |
90 |
≤150 |
95.6 |
123 |
≤200 |
no |
pass |
Results of the Dose Finding Assay
Sample |
Experiment 1 |
||
Concentration applied [µg/mL] |
Cell Viability [%] |
||
Medium Control |
-- |
-- |
96.80 |
Solvent Control |
THF |
-- |
96.60 |
Test item |
C8 |
7.81 |
95.90 |
C7 |
15.63 |
96.40 |
|
C6 |
31.25 |
96.90 |
|
C5 |
62.50 |
95.70 |
|
C4 |
125.00 |
96.10 |
|
C3 |
250.00 |
96.30 |
|
C2 |
500.00 |
96.30 |
|
C1 |
1000.00 |
96.40 |
|
Calculated CV75 [µg/mL] |
No CV75 |
||
Mean CV75 [µg/mL] |
No CV75 |
||
SD CV 75 [µg/mL] |
No SD |
||
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 |
- |
97.7 |
97.4 |
97.0 |
1487 |
894 |
556 |
931 |
338 |
89 |
97 |
267 |
161 |
Solvent Control 2 (THF) |
0.20% |
97.3 |
97.2 |
97.0 |
1354.0 |
865.0 |
553.0 |
801 |
312 |
100 |
100 |
245 |
156 |
Solvent Control 1 (DMSO) |
0.20% |
97.6 |
97.4 |
97.2 |
1577 |
876 |
527 |
1050 |
349 |
100 |
100 |
299 |
166 |
DNCB |
4.00 |
88.4 |
87.2 |
87.0 |
3792 |
1521 |
629 |
3163 |
892 |
301 |
256 |
603 |
242 |
Test item |
1000 |
96.9 |
96.9 |
97.0 |
1477 |
870 |
544 |
933 |
326 |
116 |
104 |
272 |
160 |
833.33 |
97.3 |
97.3 |
97.5 |
1415 |
860 |
534 |
881 |
326 |
110 |
104 |
265 |
161 |
|
694.44 |
97.1 |
97.1 |
97.1 |
1626 |
859 |
538 |
1088 |
321 |
136 |
103 |
302 |
160 |
|
578.70 |
96.8 |
96.5 |
96.7 |
1593 |
864 |
542 |
1051 |
322 |
131 |
103 |
294 |
159 |
|
482.25 |
96.3 |
96.1 |
96.4 |
1732 |
910 |
550 |
1182 |
360 |
148 |
115 |
315 |
165 |
|
401.88 |
95.8 |
95.8 |
95.8 |
1665 |
866 |
557 |
1108 |
309 |
138 |
99 |
299 |
155 |
|
334.90 |
96.2 |
96.6 |
96.1 |
1671 |
859 |
544 |
1127 |
315 |
141 |
101 |
307 |
158 |
|
279.08 |
96.6 |
96.7 |
95.7 |
1596 |
846 |
545 |
1051 |
301 |
131 |
96 |
293 |
155 |
|
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 |
- |
93.8 |
93.6 |
93.4 |
2804 |
1452 |
714 |
2090 |
738 |
109 |
102 |
393 |
203 |
Solvent Control 2 (THF) |
0.20% |
91.6 |
91.9 |
91.6 |
2734 |
1294 |
621 |
2113 |
673 |
100 |
100 |
440 |
208 |
Solvent Control 1 (DMSO) |
0.20% |
92.0 |
93.3 |
92.2 |
2651 |
1459 |
732 |
1919 |
727 |
100 |
100 |
362 |
199 |
DNCB |
4.0 |
76.4 |
77.2 |
75.7 |
5321 |
2804 |
680 |
4641 |
2124 |
242 |
292 |
783 |
412 |
Test item |
1000.00 |
92.20 |
92.10 |
92.00 |
1930 |
1137 |
697 |
1233 |
440 |
58 |
65 |
277 |
163 |
833.33 |
91.40 |
90.90 |
91.30 |
2142 |
1133 |
627 |
1515 |
506 |
72 |
75 |
342 |
181 |
|
694.44 |
92.60 |
92.80 |
92.70 |
2916 |
1213 |
661 |
2255 |
552 |
107 |
82 |
441 |
184 |
|
578.70 |
92.40 |
92.00 |
92.40 |
2754 |
1345 |
676 |
2078 |
669 |
98 |
99 |
407 |
199 |
|
482.25 |
91.80 |
92.10 |
91.90 |
2896 |
1304 |
655 |
2241 |
649 |
106 |
96 |
442 |
199 |
|
401.88 |
92.20 |
91.60 |
91.60 |
3092 |
1362 |
684 |
2408 |
678 |
114 |
101 |
452 |
199 |
|
334.90 |
92.20 |
92.10 |
92.70 |
2987 |
1348 |
675 |
2312 |
673 |
109 |
100 |
443 |
200 |
|
279.08 |
91.30 |
91.90 |
90.90 |
3867 |
1428 |
687 |
3180 |
741 |
151 |
110 |
563 |
208 |
|
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 |
- |
97.4 |
96.9 |
96.8 |
1948 |
889 |
633 |
1315 |
256 |
103 |
112 |
308 |
140 |
Solvent Control 2 (THF) |
0.20% |
95.7 |
96.2 |
95.6 |
1907 |
913 |
696 |
1211 |
217 |
100 |
100 |
274 |
131 |
Solvent Control 1 (DMSO) |
0.20% |
96.3 |
96.1 |
95.8 |
1925 |
872 |
643 |
1282 |
229 |
100 |
100 |
299 |
136 |
DNCB |
4.0 |
88.0 |
87.6 |
87.4 |
4413 |
1624 |
718 |
3695 |
906 |
288 |
396 |
615 |
226 |
Test item |
1000.0 |
96.6 |
96.3 |
96.1 |
2008 |
866 |
674 |
1334 |
192 |
110 |
88 |
298 |
128 |
833.33 |
96.2 |
96.4 |
96.2 |
1778 |
942 |
715 |
1063 |
227 |
88 |
105 |
249 |
132 |
|
694.44 |
96.4 |
96.5 |
96.8 |
1924 |
915 |
684 |
1240 |
231 |
102 |
106 |
281 |
134 |
|
578.70 |
95.1 |
94.9 |
94.7 |
1927 |
936 |
723 |
1204 |
213 |
99 |
98 |
267 |
129 |
|
482.25 |
95.9 |
95.9 |
95.8 |
1769 |
881 |
689 |
1080 |
192 |
89 |
88 |
257 |
128 |
|
401.88 |
96.2 |
95.9 |
95.6 |
1734 |
964 |
680 |
1054 |
284 |
87 |
131 |
255 |
142 |
|
334.90 |
96.4 |
96.7 |
96.1 |
1745 |
882 |
677 |
1068 |
205 |
88 |
94 |
258 |
130 |
|
279.08 |
96.7 |
96.8 |
96.7 |
1728 |
941 |
702 |
1026 |
239 |
85 |
110 |
246 |
134 |
|
Acceptance Criteria
Acceptance Criterion |
Range |
Exp. 1 |
pass/fail |
Exp. 2 |
pass/fail |
Exp. 3 |
pass/fail |
cell viability solvent controls [%] |
>90 |
97.0 - 97.7 |
pass |
91.6 - 93.8 |
pass |
95.6 - 97.4 |
pass |
number of test dosed with viability >50% CD86 |
≥4 |
8 |
pass |
8 |
pass |
8 |
pass |
number of test dosed with viability >50% CD54 |
≥4 |
8 |
pass |
8 |
pass |
8 |
pass |
number of test dosed with viability >50% IgG1 |
≥4 |
8 |
pass |
8 |
pass |
8 |
pass |
RFI of positive control of CD86 |
≥150 |
301 |
pass |
242 |
pass |
288 |
pass |
RFI of positive control of CD54 |
≥200 |
256 |
pass |
292 |
pass |
396 |
pass |
RFI of DMSO solvent control of CD86 |
<150 |
113 |
pass |
92 |
pass |
97 |
pass |
RFI of DMSO solvent control of CD54 |
<200 |
103 |
pass |
99 |
pass |
89 |
pass |
RFI of THF solvent control of CD86 |
<150 |
86 |
pass |
101 |
pass |
92 |
pass |
RFI of THF solvent control of CD54 |
<200 |
92 |
pass |
91 |
pass |
85 |
pass |
MFI ratio CD86/IgG1 for medium control [%] |
>105 |
267 |
pass |
393 |
pass |
308 |
pass |
MFI ratio CD86/IgG1 for DMSO control [%] |
>105 |
299 |
pass |
362 |
pass |
299 |
pass |
MFI ratio CD86/IgG1 for THF control [%] |
>105 |
245 |
pass |
440 |
pass |
274 |
pass |
MFI ratio CD54/IgG1for medium control [%] |
>105 |
161 |
pass |
203 |
pass |
140 |
pass |
MFI ratio CD54/IgG1for DMSO control [%] |
>105 |
166 |
pass |
199 |
pass |
136 |
pass |
MFI ratio CD54/IgG1for THF control [%] |
>105 |
156 |
pass |
208 |
pass |
131 |
pass |
Historical Data
Criterion |
mean |
SD |
N |
cell viability solvent controls [%] |
96.2 |
1.6 |
216 |
number of test doses with viability >50% |
- |
- |
555 |
RFI of positive control of CD86 |
358.9 |
90.1 |
36 |
RFI of positive control of CD54 |
435.7 |
285.8 |
36 |
RFI of solvent control (THF) of CD86 |
102.9 |
17.4 |
36 |
RFI of solvent control (THF) of CD54 |
102.0 |
15.4 |
36 |
MFI ratio IgG1/CD86 for medium control [%] |
285.6 |
124.3 |
36 |
MFI ratio IgG1/CD86 for THF control [%] |
270.8 |
105.0 |
36 |
MFI ratio IgG1/CD54 for medium control [%] |
308.6 |
137.1 |
36 |
MFI ratio IgG1/CD54 for THF control [%] |
158.2 |
28.4 |
36 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
The structural alerts identified in the QSAR analysis and the moderate EC3 potential for skin sensitisation estimated from the data of structural analogues provide sufficient evidence for a classification in class 1B for skin sensitisation.
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