Registration Dossier
Registration Dossier
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EC number: 285-089-6 | CAS number: 85029-63-6
Endpoint summary
Administrative data
Description of key information
The submission substance induced luciferase activity in a reliable KeratinoSens-Assay and upregulated cell surface markers in a reliable h-CLAT-Assay. Both results indicate sensitising properties.
In a DPRA, there were test item specific incompatibilities with the test method. The result of the study must be considered as inconclusive (RL3).
In a LLNA, the test item was found to be a skin sensitiser and an EC3 value of 19.3 % (w/v) was derived, indicating a moderate skins sensitising potency.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-08-01 to 2016-11-10
- 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, Draft Proposal for a new test guideline, “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)”
- 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 (345% experiment 1; 347% experiment 2) and 200%
for CD54 (496% experiment 1; 491% experiment 2) were clearly exceeded. - Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Value:
- 153
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 208
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD86 [%]
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: The 150% threshold was not exceeded at any concentration of the test item.
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: relative fluorescence intensity CD54 [%]
- Value:
- 204
- 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 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:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1.
- Executive summary:
In the present study the test item was dissolved in DMSO. A CV75 of 740.5 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:
1000.00, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90 and 279.08 µg/mL
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 93.7% (CD86), 93.6% (CD54) and 93.5% (isotype IgG1 control)in the first experiment and to 93.0% (CD86), 93.0% (CD54) and 93.6% (isotype IgG1 control)in the second experiment.
The upregulation above the threshold of 150% of the cell surface marker CD86 was observed in the first experiment only at a concentration of 578.70 µg/mL (153%). In the second experiment no upregulation above the threshold of 150% of the cell surface marker CD86 was observed. In the first experiment the expression of the cell surface marker CD54 was upregulated starting with 208% at a concentration of 578.70 µg/mL up to 268 % at the highest tested concentration (1000.00 µg/mL). In the second experiment the expression of the cell surface marker CD54 was upregulated to 204% at a concentration of 482.25 µg/mL, and 258% at a concentration of 833.33 µg/mL, up to 370% at the highest tested concentration (1000.00 µg/mL). The upregulation of the cell surface markers CD86 and CD54 were observed for both independent experiments at test item concentrations exhibiting a cell viability >50%. Since the expression of the cell surface marker CD86 exceeded the threshold in one experiment, and the cell surface marker CD54 clearly exceeded the threshold in both independent experiment the test item is considered to be a sensitiser.
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (345% experiment 1; 347% experiment 2) and 200% for CD54 (496% experiment 1; 491% experiment 2) were clearly exceeded.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-07-29 to 2016-11-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP 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 (4.90 (experiment 1); 3.64 (experiment 2); 3.97 (experiment 3)).
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: luciferase activity
- Value:
- 4.98
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: cell viability [%]
- Value:
- 119.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5 [µM]
- Value:
- 79.26
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: luciferase activity
- Value:
- 2.94
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: cell viability [%]
- Value:
- 144.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5 [µM]
- Value:
- 88.23
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: luciferase activity
- Value:
- 2.54
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: cell viability [%]
- Value:
- 111.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Parameter:
- other: EC1.5 [µM]
- Value:
- 152.23
- 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) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition which consisting of 6 wells.
The controls fullfilled the validity criteria of the test. - Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.
- Executive summary:
In the present study the test item was dissolved in DMSO. Since the test item had no defined molecular weight, the test was be performed using a pro forma molecular weight of 200 g/mol.Based on this a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. 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 4.98 was determined at a test item concentration of 1000.00 µM. The corresponding cell viability was 119.3%. The lowest tested concentration with a
significant luciferase induction >1.5 (2.05) was found to be 125.00 µM. The corresponding cell viability was <70% (62.5%).The calculated EC1.5was < 1000 µM (79.26 µM). Given that the cell viability in the lowest concentration with a significant luciferase induction >1.5 was below 70%, the prediction model was not fulfilled, and the results of experiment I where not considered for the evaluation of the sensitising potential of the test item.In the second experiment, a max luciferase activity (Imax) induction of 2.94 was determined at a test item concentration of 1000.00 µM. The corresponding cell viability was 144.9%. The lowest tested concentration with a significant luciferase induction >1.5 (1.87) was found to be 125.00 µM. The corresponding cell viability was >70% (82.1%).The calculated EC1.5 was < 1000 µM (88.23 µM).
In the third experiment, a max luciferase activity (Imax) induction of 2.54 was determined at a test item concentration of 1000.00 µM. The corresponding cell viability was 111.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.72) was found to be 250.00 µM. The corresponding cell viability was >70% (86.7%). The calculated EC1.5was < 1000 µM (152.23 µM).
A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the condition of this study the test item is therefore considered as sensitiser.
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- GLP compliance:
- yes
- Type of study:
- mouse local lymph node assay (LLNA)
- Species:
- mouse
- Strain:
- other: CBA/CaOlaHsd
- Sex:
- female
- Details on test animals and environmental conditions:
- Age: 8 - 12 weeks (beginning of acclimatization)
- Vehicle:
- dimethyl sulphoxide
- Concentration:
- The data showed that the highest test item concentration, which could be used was a 50 % solution. At this concentration the treated mice did not show any signs systemic toxicity. The test item in the main study was assayed at 10, 25, and 50 % (w/v) dissolved in DMSO.
- No. of animals per dose:
- Number of animals for each pre-test (non-GLP): 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) group: 1 - Details on study design:
- Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25 and 50 % (w/v) in DMSO. The application volume, 25 µl, was spread over the entire dorsal surface (diameter ~ 8 mm) of ear ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of 82 µCi/ml 3HTdR (corresponds to 20.5 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Determination of Incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group except the vehicle control group where only 7 nodes were taken). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Interpretation of Raw Data:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression. - Statistics:
- The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot. - Key result
- Parameter:
- EC3
- Value:
- 19.3
- Test group / Remarks:
- calculated value
- Remarks on result:
- other: positive indication of skin sensitisation
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- The test item was found to be a skin sensitiser under the described conditions.
- Executive summary:
In the study, the test item dissolved in DMSO was assessed for its possible contact allergenic potential.
For this purpose a local lymph node assay was performed using test item concentrations of 10, 25 and 50 %. The animals did not show any clinical signs during the course of the study and no cases of mortality observed. the ears of the animals treated with the high dose (50 %) were slightly red after the third application. However, no other signs of severe local irritation was observed.
In this study Stimulation Indices (S. I.) of 1.24, 4.07 and 4.41 were determined with the test item at concentrations of 10, 25 and 50 % (w/v) in DMSO, respectively.
The test item was found to be a skin sensitiser and an EC3 value of 19.3 % (w/v) was derived.
Referenceopen allclose all
Table1: Results of the Cell Batch Activation Test
Sample |
Concentration |
CD86 |
CD54 |
Activated |
||
Cell Viability [%] |
RFI |
Cell Viability [%] |
RFI |
yes/no |
||
DNCB |
4 µg/mL |
85.8 |
393 |
85.6 |
408 |
Yes |
NiSO4 |
100 µg/mL |
90.4 |
272 |
90.4 |
332 |
Yes |
LA |
1000 µg/mL |
98.1 |
56 |
98.5 |
56 |
No |
The positive controls DNCB and NiSO4led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.
The cell batch was accepted for further testing.
Table2: 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 |
0 |
96.50 |
0.00 |
97.40 |
0.00 |
96.00 |
DMSO Control |
0.00 |
96.10 |
0.00 |
97.00 |
0.00 |
98.90 |
Hostastat FE 20 LIQ |
7.81 |
96.10 |
7.81 |
97.90 |
279.08 |
98.50 |
15.63 |
96.50 |
15.63 |
97.60 |
334.90 |
98.30 |
|
31.25 |
96.30 |
31.25 |
97.30 |
401.88 |
98.20 |
|
62.50 |
95.70 |
62.50 |
97.30 |
482.25 |
98.40 |
|
125.00 |
96.00 |
125.00 |
97.30 |
578.70 |
98.30 |
|
250.00 |
96.00 |
250.00 |
96.90 |
694.44 |
98.10 |
|
500.00 |
95.00 |
500.00 |
95.40 |
833.33 |
97.30 |
|
1000.00 |
59.70 |
1000.00 |
81.80 |
1000.00 |
95.80 |
|
Calculated CV75 [µg/mL] |
740.50 |
No CV75 |
No CV75 |
|||
Mean CV75 [µg/mL] |
740.50 |
|||||
SD CV 75 [µg/mL] |
No SD |
|||||
Table3: 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 |
- |
97.2 |
97.4 |
97.4 |
1341 |
863 |
751 |
590 |
112 |
83 |
54 |
179 |
115 |
DMSO Control |
0.20% |
97.3 |
97.4 |
97.4 |
1457 |
954 |
745 |
712 |
209 |
100 |
100 |
196 |
128 |
DNCB |
4.00 |
83.7 |
84.4 |
85.3 |
3237 |
1819 |
783 |
2454 |
1036 |
345 |
496 |
413 |
232 |
Hostastat FE 20 LIQ |
1000.00 |
93.7 |
93.6 |
93.5 |
1747 |
1470 |
909 |
838 |
561 |
118 |
268 |
192 |
162 |
833.33 |
94.5 |
93.9 |
94.7 |
1862 |
1395 |
872 |
990 |
523 |
139 |
250 |
214 |
160 |
|
694.44 |
95.8 |
95.5 |
95.0 |
1901 |
1357 |
858 |
1043 |
499 |
146 |
239 |
222 |
158 |
|
578.70 |
95.4 |
95.7 |
96.1 |
1965 |
1310 |
875 |
1090 |
435 |
153 |
208 |
225 |
150 |
|
482.25 |
96.7 |
97.0 |
96.9 |
1534 |
1214 |
807 |
727 |
407 |
102 |
195 |
190 |
150 |
|
401.88 |
96.2 |
96.5 |
97.0 |
1691 |
1153 |
816 |
875 |
337 |
123 |
161 |
207 |
141 |
|
334.90 |
97.1 |
97.4 |
97.6 |
1640 |
1118 |
799 |
841 |
319 |
118 |
153 |
205 |
140 |
|
279.08 |
95.1 |
95.4 |
95.3 |
2070 |
1475 |
1118 |
952 |
357 |
134 |
171 |
185 |
132 |
|
Table4: 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 |
IgG Isotype |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
98.3 |
98.2 |
97.7 |
1351 |
885 |
713 |
638 |
172 |
85 |
106 |
189 |
124 |
DMSO Control |
0.20% |
98.0 |
97.9 |
98.2 |
1437 |
848 |
685 |
752 |
163 |
100 |
100 |
210 |
124 |
DNCB |
4.0 |
88.3 |
89.0 |
87.9 |
3363 |
1554 |
753 |
2610 |
801 |
347 |
491 |
447 |
206 |
Hostastat FE 20 LIQ |
1000.00 |
93.0 |
93.0 |
93.6 |
1863 |
1518 |
915 |
948 |
603 |
126 |
370 |
204 |
166 |
833.33 |
95.8 |
96.0 |
95.9 |
1837 |
1255 |
835 |
1002 |
420 |
133 |
258 |
220 |
150 |
|
694.44 |
97.0 |
97.4 |
96.9 |
1646 |
1155 |
850 |
796 |
305 |
106 |
187 |
194 |
136 |
|
578.70 |
97.2 |
97.7 |
97.4 |
1821 |
1124 |
819 |
1002 |
305 |
133 |
187 |
222 |
137 |
|
482.25 |
98.0 |
97.9 |
97.4 |
1663 |
1152 |
819 |
844 |
333 |
112 |
204 |
203 |
141 |
|
401.88 |
98.0 |
98.0 |
97.6 |
1596 |
1049 |
809 |
787 |
240 |
105 |
147 |
197 |
130 |
|
334.90 |
98.1 |
97.8 |
97.8 |
1518 |
1056 |
753 |
765 |
303 |
102 |
186 |
202 |
140 |
|
279.08 |
98.0 |
97.6 |
97.9 |
1642 |
1022 |
777 |
865 |
245 |
115 |
150 |
211 |
132 |
|
Table5: Acceptance Criteria
Acceptance Criterion |
Range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
||||
cell viability solvent controls [%] |
>90 |
97.2 |
- |
97.4 |
pass |
97.7 |
- |
98.3 |
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 |
345 |
pass |
347 |
pass |
||||
RFI of positive control of CD54 |
≥200 |
496 |
pass |
491 |
pass |
||||
RFI of solvent control of CD86 |
<150 |
121 |
pass |
118 |
pass |
||||
RFI of solvent control of CD54 |
<200 |
187 |
pass |
95 |
pass |
||||
MFI ratio IgG1/CD86 for medium control [%] |
>105 |
179 |
pass |
189 |
pass |
||||
MFI ratio IgG1/CD86 for DMSO control [%] |
>105 |
196 |
pass |
210 |
pass |
||||
MFI ratio IgG1/CD54 for medium control [%] |
>105 |
115 |
pass |
124 |
pass |
||||
MFI ratio IgG1/CD54 for DMSO control [%] |
>105 |
128 |
pass |
124 |
pass |
||||
Table1: Results of the Cytotoxicity Measurement
|
Concentration [µM] |
Cell Viability [%] |
||||
Experiment 1 |
Experiment 2 |
Experiment 3 |
Mean |
SD |
||
Solvent Control |
- |
100 |
100 |
100 |
100 |
0.0 |
Positive Control |
4.00 |
108.1 |
97.4 |
105.6 |
103.7 |
5.6 |
8.00 |
107.0 |
103.0 |
109.3 |
106.4 |
3.2 |
|
16.00 |
109.8 |
115.4 |
113.3 |
112.8 |
2.8 |
|
32.00 |
116.5 |
115.4 |
120.4 |
117.4 |
2.7 |
|
64.00 |
118.8 |
120.8 |
119.8 |
119.8 |
1.0 |
|
Test Item |
0.98 |
90.4 |
114.7 |
95.7 |
100.3 |
12.8 |
1.95 |
53.9 |
102.1 |
95.0 |
83.7 |
26.1 |
|
3.91 |
49.6 |
103.6 |
86.6 |
79.9 |
27.6 |
|
7.81 |
53.2 |
83.4 |
98.6 |
78.4 |
23.1 |
|
15.63 |
64.3 |
88.3 |
103.6 |
85.4 |
19.8 |
|
31.25 |
58.2 |
89.2 |
97.6 |
81.7 |
20.8 |
|
62.50 |
48.7 |
79.9 |
93.3 |
74.0 |
22.9 |
|
125.00 |
62.5 |
82.1 |
84.0 |
76.2 |
11.9 |
|
250.00 |
79.6 |
88.9 |
86.7 |
85.1 |
4.9 |
|
500.00 |
101.7 |
111.2 |
89.7 |
100.8 |
10.8 |
|
1000.00 |
119.3 |
144.9 |
111.5 |
125.2 |
17.5 |
|
2000.00 |
112.5 |
121.0 |
109.7 |
114.4 |
5.9 |
|
Table2: 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 |
1.19 |
1.27 |
1.27 |
1.24 |
0.05 |
|
8.00 |
1.39 |
1.35 |
1.34 |
1.36 |
0.03 |
|
|
16.00 |
1.85 |
1.68 |
1.39 |
1.64 |
0.23 |
* |
|
32.00 |
2.39 |
2.54 |
2.56 |
2.50 |
0.09 |
* |
|
64.00 |
4.80 |
5.58 |
4.31 |
4.90 |
0.64 |
* |
|
Test Item |
0.98 |
0.84 |
0.96 |
0.87 |
0.89 |
0.06 |
|
1.95 |
0.93 |
0.90 |
0.92 |
0.92 |
0.01 |
|
|
3.91 |
0.87 |
0.99 |
1.01 |
0.96 |
0.07 |
|
|
7.81 |
0.99 |
0.85 |
0.92 |
0.92 |
0.07 |
|
|
15.63 |
1.02 |
0.97 |
1.02 |
1.00 |
0.03 |
|
|
31.25 |
0.97 |
0.95 |
1.12 |
1.02 |
0.09 |
|
|
62.50 |
1.52 |
1.19 |
1.17 |
1.30 |
0.20 |
|
|
125.00 |
1.94 |
2.19 |
2.02 |
2.05 |
0.13 |
* |
|
250.00 |
3.15 |
3.04 |
2.54 |
2.91 |
0.33 |
* |
|
500.00 |
3.47 |
3.28 |
3.13 |
3.30 |
0.17 |
* |
|
1000.00 |
5.13 |
5.19 |
4.63 |
4.98 |
0.31 |
* |
|
2000.00 |
3.95 |
3.55 |
3.46 |
3.65 |
0.26 |
* |
|
* = significant induction according to Student’s T-test, p<0.05
Table3: 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 |
1.25 |
1.27 |
1.12 |
1.21 |
0.08 |
|
8.00 |
1.47 |
1.57 |
1.26 |
1.43 |
0.16 |
|
|
16.00 |
1.66 |
1.61 |
1.35 |
1.54 |
0.17 |
* |
|
32.00 |
1.86 |
1.95 |
1.75 |
1.85 |
0.10 |
* |
|
64.00 |
2.79 |
3.75 |
4.39 |
3.64 |
0.81 |
* |
|
Test Item |
0.98 |
1.01 |
0.87 |
1.23 |
1.04 |
0.19 |
|
1.95 |
0.98 |
0.95 |
0.73 |
0.89 |
0.14 |
|
|
3.91 |
0.95 |
0.92 |
0.90 |
0.93 |
0.03 |
|
|
7.81 |
1.01 |
0.98 |
0.95 |
0.98 |
0.03 |
|
|
15.63 |
1.00 |
1.00 |
0.93 |
0.98 |
0.04 |
|
|
31.25 |
1.06 |
1.06 |
1.05 |
1.06 |
0.01 |
|
|
62.50 |
1.17 |
1.39 |
1.17 |
1.24 |
0.12 |
|
|
125.00 |
1.90 |
2.17 |
1.53 |
1.87 |
0.32 |
* |
|
250.00 |
2.28 |
2.54 |
2.05 |
2.29 |
0.24 |
* |
|
500.00 |
2.55 |
2.69 |
2.41 |
2.55 |
0.14 |
* |
|
1000.00 |
3.15 |
2.84 |
2.81 |
2.94 |
0.19 |
* |
|
2000.00 |
2.57 |
2.91 |
2.91 |
2.80 |
0.20 |
* |
|
* = significant induction according to Student’s T-test, p<0.05
Table4: Induction of Luciferase Activity Experiment 3
Experiment 3 |
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 |
1.42 |
1.18 |
1.29 |
0.13 |
|
8.00 |
1.34 |
1.21 |
1.18 |
1.24 |
0.09 |
|
|
16.00 |
1.86 |
2.01 |
1.83 |
1.90 |
0.10 |
* |
|
32.00 |
2.16 |
1.79 |
2.23 |
2.06 |
0.24 |
* |
|
64.00 |
3.94 |
4.02 |
3.95 |
3.97 |
0.04 |
* |
|
Test Item |
0.98 |
0.92 |
1.34 |
1.04 |
1.10 |
0.21 |
|
1.95 |
0.96 |
1.13 |
1.01 |
1.04 |
0.09 |
|
|
3.91 |
0.78 |
1.09 |
1.00 |
0.96 |
0.16 |
|
|
7.81 |
0.84 |
0.93 |
0.99 |
0.92 |
0.07 |
|
|
15.63 |
0.86 |
0.94 |
0.90 |
0.90 |
0.04 |
|
|
31.25 |
0.97 |
1.15 |
1.13 |
1.08 |
0.10 |
|
|
62.50 |
0.85 |
1.18 |
1.09 |
1.04 |
0.17 |
|
|
125.00 |
1.33 |
1.54 |
1.45 |
1.44 |
0.11 |
|
|
250.00 |
1.48 |
1.89 |
1.78 |
1.72 |
0.21 |
* |
|
500.00 |
1.88 |
2.31 |
1.83 |
2.01 |
0.27 |
* |
|
1000.00 |
2.31 |
2.72 |
2.59 |
2.54 |
0.21 |
* |
|
2000.00 |
2.23 |
1.93 |
2.06 |
2.07 |
0.15 |
* |
|
Discussion
In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 10, 25 and 50 % (w/v) in DMSO by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (DMSO) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.
All treated animals survived the scheduled study period.
A test item is regraded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3 -fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S. I.). The estimated concentration of test item required to produce a S. I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 1.24, 4.07 and 4.41 were determined with the test item at concentrations of 10, 20 and 50 % (w/v) in DMSO revealing a clear dose-response relation. The EC3 value calculated was 19.3 % (w/v) pointing to a moderate potency.
In the OECD Test Guideline it is stated that a false positive outcome might results for some surfactant type chemicals, i.e. using a positive result from an LLNA performed with a surfactant might be an overestimation of the real potency. Although the test item has surface active properties it is assumed that the test is valid for the submission substance, as the components of the submission substance do not have any unsaturated bindings, which are considered responsible for the false positive outcomes in the LLNA (Kreiling et al. 2008: Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT). Food Chem. Toxicol. 2008, 46: 1896-1904). Therefore, the result of the LLNA is used for the derivation of the skin sensitising potency of the submission substance.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
In theKeratinoSens-Assay, the results of experiment did not fulfil the prediction model and where not considered for the evaluation of the sensitising potential of the test item.
In the second experiment, a max luciferase activity (Imax) induction of 2.94 was determined at a test item concentration of 1000.00 μM. The corresponding cell viability was 144.9%. The lowest tested
concentration with a significant luciferase induction >1.5 (1.87) was found to be 125.00 μM. The corresponding cell viability was >70% (82.1%).The calculated EC1.5 was < 1000 μM (88.23 μM).
In the third experiment, a max luciferase activity (Imax) induction of 2.54 was determined at a test item concentration of 1000.00 μM. The corresponding cell viability was 111.5%. The lowest tested
concentration with a significant luciferase induction >1.5 (1.72) was found to be 250.00 μM. The corresponding cell viability was >70% (86.7%).The calculated EC1.5was < 1000 μM (152.23 μM).
A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as sensitiser.
In the h-CLAT-Assay, the upregulation of the cell surface markers CD86 and CD54 were observed for both independent experiments at test item concentrations exhibiting a cell viability >50%. Since the expression of the cell surface marker CD86 exceeded the threshold in one experiment, and the cell surface marker CD54 clearly exceeded the threshold in both independent experiment the test item is considered to be a sensitiser.
DPRA:Due to a strong interference of the test item with the HPLC no results about the lysine peptide depletion were available and prediction model 2 based on the cysteine peptide only should be considered. The observed phase separation in the samples containing cysteine peptide and test item is considered as test item specific incompatibility with the test method and a second run was considered as not feasible. Hence, a prediction of the sensitising potential cannot be made. Due to the observed phase separation the mean peptide depletion of the cysteine peptide may reflect an underestimation of the reactivity.
LLNA: Stimulation Indices (S. I.) of 1.24, 4.07 and 4.41 were determined with the test item at concentrations of 10, 25 and 50 % (w/v) in DMSO, respectively. The test item was found to be a skin sensitiser and an EC3 value of 19.3 % (w/v).
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
According to the available in vitro and in vivo data, the submission substance has to be classified as skin sensitising. Based on the potency estimate derived from the LLNA (EC3: 19.3%) a classification as skin sensitising Cat. 1B according to Regulation (EC) No. 1272/2008 (CLP) is recommended.
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