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Diss Factsheets

Administrative data

Description of key information

Not a skin sensitiser

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 14 to March 03, 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM (INVITTOX) Protocol n°155: KeratinoSens™
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
FORMULATION OF TEST ITEM
Test item could not be dissolved in DMSO after approximately 5 minutes of vortexing, but formed heterogeneous heavy flowing suspension. In ultrapure water, a partial dissolution of test item could be observed with undissolved particles floating in the solution. Thus, a less concentrated 100 mM solution was tried, with additional volume of ultrapure water a free flowing dark solution was gained. Since the overall volume of the above mentioned formulation did not allow proper visual inspection of homogeneity and was not sufficient for sterile filtering, a 10× volume was prepared at the same 100 mM concentration. This way a homogenous, free flowing solution was gained after 5 minutes of vortexing and was suitable for sterile filtration (through 0.22 micron filter membrane).
Since the formula fulfilled all requirements ultrapure water was chosen as the appropriate solvent of the test item.

CELLS USED
- Cell line: KeratinoSens™ cell line
- Description: immortalized adherent cell line derived from human keratinocytes (HaCaT) transfected with selectable plasmids.
- Supplier: Givaudan Schweiz AG
- Storage: vapor phase of liquid nitrogen.
- Subculturing: the original cells were propagated and subcultured into prepared cell line stocks (master cultures - MCs) in testing laboratory. Cells from this original stock could be propagated up to maximum 25 passages and were employed for routine testing using the maintenance/growth medium.

TEST CHEMICAL MASTER SOLUTION
Based on the test chemical stock solution made with ultrapure water at the highest soluble concentration (100 mM), two fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test chemical creating a 100 × master plate. The 100 × master concentrations were further diluted 25 fold into exposure medium to obtain the 4 × master plate, by adding 10 µl of the 100 × master concentrations to 230 µl exposure medium and correcting DMSO concentration by adding 10 µl DMSO to all twelve 4 × master solutions.

POSITIVE CONTROL
Trans-cinnamaldehyde, dissolved in Dimethyl sulfoxide (DMSO). A series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 µM.

NEGATIVE CONTROL
The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates.

PREPARATION OF THE CELLS
For testing cells were 80 - 90 % confluent and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium, and distributed into 96-well plates (10000 cells/well) homogenously; no sedimentation of the cells occurred during seeding. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.

EXPOSURE
After the 24 hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test chemical and control substances were added to each well in a way that an additional 4 fold dilution was achieved on the plate for the final concentrations to be established (50 µl of 4× master solution to 150 µl of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test chemical.

LUCIFERASE ACTIVITY MEASUREMENTS
After the 48 hour exposure time with the test chemical and control substances, cells were washed with DPBS (270 µl), and 1× lysis buffer (20 µl) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 µl) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

CITOTOXICITY
For the cell viability assay, medium was replaced after the 48 hour exposure time with MTT working solution (200 µl) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilized by the addition of isopropanol (50 µl). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

ACCEPTANCE CRITERIA
For each test chemical and positive control substance, in order to derive a prediction, at least two independent tests, each containing three replicates for the luminescence measurements and one for viability measurement, were needed. In case of discordant results between the two independent tests, a third test containing four replicates should be performed. Each independent test was to be performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may have come from the same passage however. KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the limitations stated in the OECD test guideline.

The luciferase activity induction obtained with the positive control, trans-cinnamaldehyde should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility or between 7 μM and 30 μM (based on the validation dataset). In addition, the average induction in the parallel plates for trans-cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of trans-cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Historical control mean for EC1.5 value of the positive control: 14.6 ± 9.1 μM
Acceptance range for EC1.5 value of the positive control: 5.6 μM – 23.7 μM

Finally, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO should be below 20 % in each test which consists of 6 wells tested in triplicate.
Run / experiment:
other: experiment 1
Parameter:
other: Imax
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: experiment 2
Parameter:
other: Imax
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
There was no statistically significant induction above 1.5-fold observed at any of the concentrations in either of the tests; therefore, no EC1.5 values were determined for the test item. The maximal fold induction was 1.29 for the first test and 1.34 for the second test. Based on the prediction model and the above described results, both tests were concluded negative.

There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at several concentrations in both tests. Cells of the last three wells were stained by the higher concentrations of the dark purplish - bluish test item solutions thus the resulted dark color may have affected absorbance measurement, and might gave a false sense of high survival rates. In the first test, the IC30 and IC50 values were 26 µM and 50 µM while in the second test 29 µM and 58 µM, respectively.

NEGATIVE CONTROL
The coefficient of variation (CV %) of the luminescence reading for the negative control DMSO was below 20 % in either of the tests (18.17 % and 18.86 % respectively). However one well of negative control (DMSO) values was omitted from the calculation as an outlier, since it is 25 % greater than the average of the other 5 values on the same plate in the second test.

POSITIVE CONTROL
The luciferase activity induction obtained with the positive control, trans-cinnamaldehyde was statistically significant above the threshold of 1.5 at four concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 6 µM and 9 µM in the two individual tests which fell into the corresponding acceptance range established based on the historical control data: 5.6 μM – 23.7 μM.
In addition, the average induction in the parallel plates for trans cinnamaldehyde at 64 μM was 28.35 and 83.88 fold. Although these values are out of the 2 – 8 fold induction range, the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control, therefore the tests were considered valid.

There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations in the first test however, there was cytotoxicity (viability < 70 %) induced by the positive control at 32 and 64 µM in the second test.

Average fold induction, significance and viability (%) values for the test item in the individual tests

Experiment 1

Concentration (uM) 0.5 1 2 4 8 16 31 63 125 250 500 1000
Plate 1 0.93 1.38 1.19 1.12 0.83 0.79 0.79 1.51 0.00 -0.02 -0.02 -0.02
Plate 2 1.21 1.13 1.5 1.01 0.9 0.74 0.74 1.33 0.00 -0.02 -0.02 -0.02
Plate 3 0.81 1.12 1.19 0.9 0.72 0.61 0.56 0.93 0.04 0.00 -0.01 -0.01

average induction

0.98

1.21

1.29 1.01 0.82 0.71 0.7 1.26 0.01 -0.02 -0.02 -0.02

significance

0.917 0.034 0.136 0.987 0.102 0.013 0.015 0.141 0.000 0.000 0.000 0.000

viability*

95 % 85 % 91 % 80 % 79 % 81 % 65 % 40 % 6 % 48 % 84 % 85 %

*Cells of the last three wells were stained by the higher concentrations of the test item solutions thus the resulted purple color may have affected absorbance measurement

Experiment 2

Concentration (uM)

0.5 1 2 4 8 16 31 63 125 250 500 1000
Plate 1 1.36 1.51 1.35 1.35 1.65 0.89 1.03 1.79 0.00 -0.03 -0.03 -0.03
Plate 2 1.06 1.03 1.03 0.81 0.81 0.35 0.58 1.31 -0.02 -0.06 -0.06 -0.06
Plate 3 0.89 0.83 1.01 1.01 1.29 0.57 0.68 0.92 -0.03 -0.05 -0.05 -0.05

average induction

1.10 1.12 1.13 1.05 1.25 0.61 0.76 1.34 -0.02 -0.04 -0.05 -0.05

significance

0.514 0.548 0.372 0.829 0.383 0.056 0.118 0.129 0.001 0.001 0.001 0.001

viability*

130 % 141 % 163 % 126 % 112 % 102 % 66 % 47 % 13 % 179 % 248 % 190 %

*Cells of the last three wells were stained by the higher concentrations of the test item solutions thus the resulted purple color may have affected absorbance measurement

Interpretation of results:
other: test result is evaluated as part of a weight of evidence
Conclusions:
Based on the KeratinoSens™ prediction model, the test item was concluded negative, therefore, having a non-sensitizing potential under the experimental conditions.
Executive summary:

In the course of the study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test chemical and positive control substance, two independent tests were sufficient to derive a prediction, as test results were concordant and both tests met the acceptance criteria.

The luciferase activity induction obtained with the positive control, trans-cinnamaldehyde was statistically significant above the threshold of 1.5 at four concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 6 µM and 9 µM in the two individual tests respectively. In addition, the average induction in the parallel plates for trans cinnamaldehyde at 64 μM was 28.35 and 83.88 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations in the first test; however, there was cytotoxicity (viability < 70 %) induced at 32 and 64 µM in the second test.

For the test item twelve doses were used in two independent tests between 1000 µM to 0.5 µM.  There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at concentrations starting from 31 µM in both tests. In the first test, IC30 and IC50 values were 26 µM and 50 µM, while in the second test 29 µM and 58 µM respectively. However, the fold induction did not exceed the threshold of 1.5-fold at any concentrations compared to the respective negative controls in any of the independent tests. Therefore, both tests were concluded negative for luciferase gene induction.

Conclusion

Based on the KeratinoSens™ prediction model, test item was concluded negative, therefore, having a non-sensitizing potential under the experimental conditions.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From October 12 to November 05, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Details on the study design:
FORMULATION OF TEST ITEM
- Vehicle: sodium chloride solution (saline)
- Reason of chioce: solubility of the test item was evaluated in sodium chloride solution (saline) at the concentration of 100 mg/ml; in saline at the concentration of 100 mg/ml the test item formed a dark blue, homogenous and free-flowing solution.

CONTROLS
- Positive control: 1-chloro-2,4-dinitrobenzene.
- Negative control: sodium chloride solution.
- Solvent control: dimethyl sulfoxide (DMSO).

CELL
- Cells: THP-1; the THP-1 cell line is an immortalized human monocytic leukemia cell line.
- Supplier: ATCC.
- Maintenance (culture) medium: RPMI-1640 modified medium (with 25 mM HEPES buffer) supplemented with 10 (v/v) % fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.05 mM L glutamine solution and 0.05 mM 2 mercaptoethanol.

CONCENTRATIONS: 349, 419, 502, 603, 723, 868, 1042 and 1250 µg/ml.
The test item was first diluted to the concentration corresponding to the highest soluble concentration determined in the dose finding assay. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using saline (8 concentrations). The master solutions were then further diluted 50-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate.

TEST SYSTEM - MAIN TEST
- Application of test item and control substances: test item and control substances were mixed with 500 μl of suspended cells (1 × 10^6 cells) at 1:1 ratio in a single replicate.
- Incubation: 24 ± 0.5 hours at 37 °C under 5 % CO2.

FITC STAINING
After 24 ± 0.5 hours of exposure, cells were transferred from 24-well plate into sample tubes, then 1 ml of FACS buffer was added to each sample and cells were collected by centrifugation (250 g, 5 min, 4 ºC). The washing step was repeated once more with 1 ml of FACS buffer. After washing, cells were blocked with 600 μl of 1 × blocking solution and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots of 200 μl into sample tubes.
After centrifugation (250 g, 5 min, 4 ºC), cells were stained with 50 μl of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at 4 °C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μl of FACS buffer, cells were resuspended in 400 μl of FACS buffer and 20 μl of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

PRELIMINARY TESTS
- Preparation of the cells: THP-1 cells were seeded at a density of either 0.1 × 10^6 cells/ml or 0.2 × 10^6 cells/ml and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintance medium at 2 × 10^6 cells/ml. Then, cells were distributed into 24 well flat-bottom plate with 500 µl / wells (1 × 10^6 cells/well).
- Master and working solutions: 8 master solutions (8 concentrations) were prepared of the test item stock solution, by two-fold serial dilutions using saline. These master solutions were then further diluted 50 fold into culture medium to obtain the working solutions (WS). The working solutions were finally used for exposure by adding an equal volume of working solution (500 µl) to the volume of THP-1 cell suspension (500 µl) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item.
- Test item exposure: the culture medium or working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well.
- Incubation: the treated plates were then incubated for 24 ± 0.5 hours at 37 °C under 5 % CO2. The plates were sealed with breathable microplate covers prior to the incubation to avoid evaporation of test item.
- Replicate: 2 independent runs.
- Concentrations: 8, 16, 31, 63, 125, 251, 502 and 1003 µg/ml and 10, 20, 39, 78, 157, 313, 627 and 1253 µg/ml.
- PI Staining: cells were transferred into sample tubes and 600 μl of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μl of FACS buffer. Finally, cells were resuspended in 400 μl of FACS buffer and 20 μl of 1 × PI solution was added for each sample.
- Cytotoxicity measurement by flow cytometry and estimation of CV75 value: the PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10000 living cells (PI negative) were acquired.

ACCEPTANCE CRITERIA
Requirements for qualified testing
- The cell viabilities of medium and solvent/vehicle controls should be higher than 90 %.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 and CD54 ≥ 200) and cell viability should be more than 50 %.
- In the solvent controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 > 150 and CD54 > 200).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105 %.

Abnormal values
- RFI values cannot be less than zero. Regardless of the reason, such values should be omitted from the prediction.
- If an abnormal value is observed, check whether there are abnormal conditions in the run and record them in the reporting section.

Requirement for data acceptance
- For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90 % (when HSC is used instead of CV75, the data for the test chemical is accepted regardless the cell viability at the highest dose.
- For the test item, the cell viability should be more than 50 % in at least four tested concentrations in each run.
Run / experiment:
other: 2/2 runs
Parameter:
other: Relative Fluorescence Intensity of CD86 (%)
Value:
150 %
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 2/2 runs
Parameter:
other: Relative Fluorescence Intensity of CD54 (%)
Value:
200 %
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
For the test item, the relative fluorescence intensity (RFI) of CD86 was lower than 150 % at all tested doses (with cell viability > 50 %) in both runs.
The RFI values for CD54 expression were lower than 200 % at all tested doses (with cell viability > 50 %) in both runs.
Since RFI values cannot be less than zero, in the first run the RFI value of CD86 at 1041 µg/ml was omitted from the prediction.
Since 2 out of 2 runs were negative for CD86 and CD54 marker expressions, the overall outcome of the study was negative.

CONTROLS
Both runs were considered valid, since both runs have met the acceptance criteria.
The positive control gave positive results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50% in each run.
The DMSO controls had negative outcomes compared to the medium control for both markers in all runs, meaning that the RFI values of both CD86 and CD54 expression was never over the positive criteria.
The cell viabilities of medium and DMSO controls were higher than 90 % in all runs (taken cell viabilities of the IgG1 isotypic control). For medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control was over 105 %.

PRELIMINARY TEST - Dose finding assays
Cell viability did not lower to 75 % at any tested concentrations in either of the runs. Since not even the highest soluble concentration caused significant reduction in cell viability, no CV75 value could be determined. Therefore, the highest soluble stock concentration (125 mg/ml) was used for setting the dose-range for measuring CD86 and CD54 expression in the main test.

Results on test item

Conc. (µg/ml) MFI (geo mean) RFI (CD86) RFI (CD54) IgG viability (%)
CD86 CD54 Isotype
First run
Control 2967 3344 2427 100 100 92.3
DMSO 0.2 % 2498 2752 2168 61 64 93.5
DNCB, 4.2 13320 5444 24056 3308 520 66.0
Test item, 1252 1715 1690 1352 65 36 50.4
Test item, 1043 1452 1760 1528 -14 25 58.9
Test item, 869 1833 2053 1647 34 44 65.2
Test item, 725 1940 2175 1848 17 36 67.4
Test item, 604 2042 2352 1812 43 59 61.9
Test item, 503 2090 2191 1891 37 33 62.2
Test item, 419 2059 2355 1870 35 53 67.5
Test item, 349 2143 2352 2040 19 34 66.6
Second run
Control 4243 3052 2311 100 100 94.0
DMSO 0.2 % 4227 2743 2141 108 81 94.6
DNCB, 4.2 12183 4831 2394 469 405 80.5
Test item, 1252 1967 2069 1852 6 29 71.9
Test item, 1043 2006 2186 1834 9 48 76.2
Test item, 869 1952 2347 1901 3 60 73.1
Test item, 725 2000 2549 1825 9 98 74.1
Test item, 604 2148 2714 1754 20 130 80.2
Test item, 503 1769 2719 1676 5 141 86.8
Test item, 419 2108 2503 1705 21 108 90.7
Test item, 349 2325 2667 1754 30 123 89.7

Positive and negative control data

Sample Concentration RFI  viability (IgG1)
CD86 CD54 IgG
Replicate 1 DMSO 0.20 % 61 64 93.5
DNCB 4.2 μg/ml 3308 520 66.0
Replicate 2 DMSO 0.20 % 108 81 94.6
DNCB 4.2 μg/ml 469 405 80.5
Interpretation of results:
other: test result is evaluated as part of a weight of evidence
Conclusions:
The test item was concluded negative and demonstrated a non-sensitizing potential under the experimental conditions of human Cell Line Activation Test.
Executive summary:

The skin sensitization potential of the test item was studied. The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. CV75 could not be determined, since there was no test item concentration that resulted in 75 % cell viability compared to the solvent/vehicle control. Thus, the highest soluble concentration (125 mg/ml) was determined and used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in two independent runs between 1252 – 349 µg/ml.

The increase in CD86 marker expression (RFI) was not equal to or greater than 150 % at any tested dose (with >50 % of cell viability) compared to the respective negative controls in any of the independent runs. Therefore, both runs were negative for CD86 marker expression.

Also, the increase in CD54 marker expression (RFI) was not equal to or greater than 200 % at any tested dose (with >50 % of cell viability) compared to the respective negative controls in any of the independent runs. Based on the concordant negative results of the two runs, CD54 marker expression was not induced by the test item.

Since none of the markers gave positive result, the overall h-CLAT prediction was concluded negative, as well.

Conclusion

Based on the results and the h-CLAT prediction model, the test item was concluded negative and demonstrated a non-sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Two in vitro experimental studies were available which assessed the sensitising potential of the test item. Specifically, these two studies examine the ability of the test item to induce a keratinocyte response (AOP Key Event 2, in an ARE Nrf2 LuSens assay) and a monocytic/dendritic cell response (AOP Key Event 3, in a h-CLAT). The ability of the substance to induce peptide/protein bonding (Adverse Outcome Pathway (AOP) Key Event 1 is assessed in a DPRA, which is not compatible with the substance due to structural limitations, however, the two negative results are conclusive for no sensitisation potential. The following experimental findings are reported:

(1) The test item's potential to activate the Nrf2 transcription factor was assessed in the genetically modified keratinocyte cell-line ARE Nrf2 Luciferase “LuSens” Assay (Bauch et al. 2012), according to the OECD guideline 442D (2017). No substantial, reproducible, dose-dependent increase in luciferase induction above 1.5-fold was observed up to the maximal test item concentration of 2000 µg/ml. Therefore, the assay was negative and the test item is not considered to have the potential to activate Nrf2 transcription factor and does not induce a keratinocyte response.

(2) A Human Cell Line Activation Test (h-CLAT) was performed on the human monocytic leukaemia cell line (THP-1 cells), according to the OECD guideline 442E (2018). None of the experiments produced positive results, therefore, the test item is not considered to have the potential to activate a dendritic cell response.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008, a skin sensitiser is an agent that will lead to an allergic response in susceptible individuals following skin contact. As a consequence of a secondary - usually organ-specific - subsequent re-exposure, adverse health effects occur on the skin (allergic contact dermatitis). Skin sensitisers are classified in Category 1 - H317. Where data is sufficient, skin sensitisers can be divided into sub-categories. If data are not sufficient for sub-categorisation, Category 1 must be chosen. The CLP (and UN GHS) criteria for classifying sensitisers are based on standard animal data and human data; data obtained from non-standard methods such as read-across or in vitro/in chemico test methods may be used in combination in a Weight of Evidence approach.

Indicators of potency of a substance can be obtained from in chemico/in vitro testing; specifically, the following tests may be accepted to fulfill the requirements of Annex VII:

(i) Direct Peptide Reactivity Assay (DPRA) addresses AOP Key Event 1: Peptide/protein binding

(ii) ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) addresses AOP Key Event 2: Keratinocyte response

(iii) the Human Cell Line Activation Test (h-CLAT) addresses AOP Key Event 3: Monocytic /Dendritic cell response.

These test methods were developed to address specific events of the skin sensitisation AOP (OECD, 2012). The AOP for skin sensitisation describes the current understanding of key events linked to skin sensitisation. As each of the test methods only addresses a specific key event of skin sensitisation, currently they should not be used in isolation to identify a potential skin sensitiser but rather in combination in a Weight of Evidence approach.

The DPRA could not be performed due to structural incompatibility. Based on the negative LuSens assay (inflammatory response in keratinocytes) and negative h-CLAT (dendritic cell activataion), the data is sufficient for a Weight of Evidence approach as requirements for Article 13(3) are fulfilled. Using a Weight of Evidence approach, test item can be considered not potentially skin sensitising, according to the CLP Regulation (EC 1272/2008).