Registration Dossier

Administrative data

Description of key information

Skin sensitisation: not sensitising based on weight of evidence from 3 in chemico/in vitro studies.

- positive in DPRA (OECD 442C, GLP)

- negative in KeratinoSens (OECD 442D, GLP)

- negative in U-SENS (OECD 442E, GLP)

Respiratory sensitisation: no study available

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-24 to 2018-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
Test system: Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

Number of replicates: co-elution controls, reference controls and samples were measured in triplicates.
SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. In addition, a RCcysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL MQ.

Co-elution control (CC): 750 µL Phosphate buffer pH 7.5, 200 µL ACN, 50 µL CCys 209629/A solution (100 mM)

Positive control: Cinnamic aldehyde (PC): 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL Cinnamic aldehyde solution (100 mM in ACN).

Test item 209629/A: 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN, 50 µL 209629/A test solution (100 mM)
SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN. In addition, a RClysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL MQ.

Co-elution control (CC):750 µL Ammonium acetate buffer pH 10.2, 250 µL CClys 209629/A test solution (100 mM)

Positive control: Cinnamic aldehyde (PC): 750 µL Stock solution of 0.667 mM SPCL, 250 µL Cinnamic aldehyde solution (100 mM in ACN)

Test item 209629/A: 750 µL Stock solution of 0.667 mM SPCL, 250 µL 209629/A test solution (100 mM)

After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 23.5 hours and 25 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC PDA analysis the samples were visually inspected for precipitation.
At a concentration of 100 mM, TA-1 was not soluble in ACN, but was soluble in MQ. Therefore this solvent was used to dissolve the test item in this DPRA study. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.
SPCC and SPCL peak areas in the samples were measured by HPLC PDA.
Mobile phase: A: 0.1% (v/v) TFA in Milli-Q water, B: 0.085% (v/v) TFA in ACN
Flow:0.35 mL/min
Injection volume: 3 µL
Sample tray temperature: Set at 25°C
Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 µm (Agilent Technologies, Santa Clara, CA, USA)
Guard column: SecurityGuard™ cartridge for C18, 4 x 2.0 mm (Phenomenex, Torrance, CA, USA)
Column temperature: Set at 30°C
Detection: Photodiode array detection, monitoring at 220 and 258 nm
Positive control results:
% depletion cystein peptide: mean 68.9%; SD 0.3%
% depletion lysine peptide: mean 60.5%; SD 2.1%
Key result
Parameter:
other: mean percent cysteine and lysine depletion
Value:
13.5
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Remarks:
low reacticity
Key result
Parameter:
other: mean percent depletion
Run / experiment:
cysteine peptide
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: mean percent depletion
Run / experiment:
lysine peptide
Value:
27
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
low reactivity
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

Acceptability of the Cysteine Reactivity Assay
The correlation coefficient (r²) of the SPCC standard calibration curve was 0.998. Since the r² was >0.99, the SPCC standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.518 ± 0.005 mM, the mean peptide concentration of Reference Controls C was 0.518 ± 0.006 mM and the mean peptide concentration of Reference Controls CMQ was 0.506 ± 0.002 mM. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent SPCC Depletion. The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 18.00. The mean A220/A258 ratio ± 10% range was 16.20-19.80. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 68.9% ± 0.3%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the Lysine Reactivity Assay
The correlation coefficient (r²) of the SPCL standard calibration curve was 0.998. Since the r² was >0.99, the SPCL standard calibration curve was accepted.The mean peptide concentration of Reference Controls A was 0.511 ± 0.018 mM, the mean peptide concentration of Reference Controls C was 0.532 ± 0.013 mM and the mean peptide concentration of Reference Controls CMQ was 0.500 ± 0.016 mM. The means of Reference Control samples A, C and CMQ were all within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve the test item did not impact the Percent SPCL Depletion. The CV of the peptide areas for the nine Reference Controls B and C was 2.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A220/A258) of the Reference Control samples was 16.85. The mean A220/A258 ratio ± 10% range was 15.17-18.54. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.5% ± 2.1%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

The test item was found not soluble at 100 mM in acetonitrile. A solution was obtained at 100 mM with MQ water.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

 

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide.

The mean of the percent cysteine and percent lysine depletions was equal to 13.5%. Accordingly, the test item was considered to have low peptide reactivity. Therefore, the DPRA prediction would be considered as positive and the test item may have a potential to cause skin sensitization.

 

Interpretation of results:
other: positive
Conclusions:
In conclusion, the test item was tested for skin sensitisation in the Direct Peptide Reactivity Assay. This DPRA test is considered valid, thus, the test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-24 to 2018-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™)
- Details on study design:
Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium. All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 83 - 99%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 concentration was monitored once on each working day. Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25). For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+11 in experiment 1 and P+13 in experiment 2.
Treatment of Cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 h ± 1 h at 37±1.0°C in the presence of 5% CO2. In total 2 experiments were performed. In the main experiments the test item was suspended in DMSO at 200 mM (white suspension). The stock was sonicated (Time: 18 - 30 min; Temp.: 20 - 35°C). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate.
Luciferase Activity Measurement :
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the luciferase activity (integration time two seconds).
Viability measurement:
For the KeratinoSens™ cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37±1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
Acceptability criteria:
The KeratinoSens™ test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant above the threshold of 1.5 (e.g. using a t-test) in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 of the positive control should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol 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.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test (2). If the variability is still higher, the results should be discarded.
Controls:
A blank, a vehicle control and a positive control were set up in parallel in order to confirm the validity of the test.
Blank:
On each plate three blank wells were tested (no cells and no treatment).
Vehicle control:
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.
Positive control:
The positive control used in the case of KeratinoSens™ is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.
Data analysis:
The following parameters are calculated in the KeratinoSens™ test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Prediction model:
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
1. The Imax is equal or higher than (≥) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity ≥ 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 µM or 200 µg/mL and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should be considered as inconclusive.
Positive control results:
The positive control (EDMG) values are considered to be valid because they are within the acceptability range demanded by the OECD test guideline 442d and the determined historical data (see also Tables 1 & 2).
Key result
Parameter:
other: Luciferase activity Imax
Remarks:
[fold increase]
Run / experiment:
1
Value:
1.25
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Luciferase activity Imax
Remarks:
[fold increase]
Run / experiment:
2
Value:
1.15
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Toxicity IC30
Remarks:
[µM]
Run / experiment:
1 and 2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Parameter:
other: Toxicity IC50
Remarks:
[µM]
Run / experiment:
1 and 2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Parameter:
other: Luciferase activity EC1.5
Remarks:
[µM]
Run / experiment:
1 and 2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO is below 20% in each repetition which consists of 18 wells tested
- Acceptance criteria met for positive control: Yes, EC1.5 is within two SD of the historical mean: 36 µM in experiment 1 and 39 µM in experiment 2.

Table 2: Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp 1 luminescence

1.21

1.38

1.46

1.71***

2.08***

3.12***

Exp 1 viability (%)

101.3

106.7

112.3

119.6

121.1

128.3

Exp 2 luminescence

1.28

1.31

1.44

1.67***

1.90***

2.43***

Exp 2 viability (%)

101.7

102.5

104.3

116.7

120.9

133.6

***p<0.001 Student’s t test

Table 3: Overview Luminescence Induction and Cell Viability of the test item in Experiment 1 and 2

Concentration (µM)

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

2000

Exp 1 luminescence

1.02

1.10

1.14

1.24

1.24

1.25

1.20

1.21

1.22

1.12

1.10

1.00

Exp 1 viability (%)

99.7

91.1

88.6

86.9

85.2

84.9

84.4

87.3

85.8

91.8

89.9

92.9

Exp 2 luminescence

0.94

1.04

1.11

1.15

1.09

1.09

1.04

1.06

1.00

0.96

0.71

0.72

Exp 2 viability (%)

104.7

107.1

94.3

84.0

84.0

81.7

80.5

85.3

84.5

89.3

98.8

106.4

Interpretation of results:
other: negative
Conclusions:
The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.25-fold and 1.15-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSens™ assay since negative results (< 1.5-fold induction) were observed at test concentrations up to 2000 µM.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-07-26 to 2018-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
Stock cultures of U937 human monocytes are stored in the freezer (-150°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test.
Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.
Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested. Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL.
In the second experiment cells were treated with four selected doses of test item. At least
2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 20, 50, 100 and 200 µg/mL.
In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.
After 45 h cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells. FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.
Acquisition
Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
Analysis
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot.
The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity were analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.

ACCEPTABILITY CRITERIA
• At the end of the incubation treatment period, the mean viability of the triplicate untreated (RPMI) U937 cells is > 90%
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
• No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected %CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
• The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
• Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%).

If (one of) the acceptability criteria are not met and the Study Director decides that this has a critical effect on the study, the test was rejected and repeated.
INTERPRETATION
• For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
• The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 X Median S.I. ≥ 150%).
• In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
• An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N) (Figure 1). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
• An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P) (Figure 1). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
• There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4).
Positive control results:
The positive control exhibited a stimulation factor ≥ 676% and ≥ 358% in all wells and showed no cytotoxicity in experiment 1 and 2, respectively.
Key result
Parameter:
other: CV70 (cytotoxicity)
Remarks:
[µg/mL]
Run / experiment:
1 and 2
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
CV70 could not be detected under the present conditions
Key result
Parameter:
other: EC150 (induction of CD86 activity)
Remarks:
[µg/mL]
Run / experiment:
1 and 2
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
An EC150 could not be detected under the present conditions
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%). Experimenmt 1 and 2: No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with TA-1. No EC150 could be calculated and is considered to be higher than 200 µg/mL.
- Acceptance criteria met for positive control: The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%). Experiment1: The positive control (TNBS) showed a S.I. ≥ 676% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 96% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). Experiment 2: The positive control (TNBS) showed a S.I. ≥ 358% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 118% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control

Controls

% Viability (Mean)*

CD86-IgG1 S.I.*

Experiment

Experiment

1

2

1

2

LA1

99

99

96

109

LA2

99

99

76

103

LA3

99

98

76

118

TNBS1

98

99

730

358

TNBS2

98

98

846

430

TNBS3

99

98

676

442

 

IgG1 value (%)

CD86 basal expression (%)

Experiment

Experiment

1

2

1

2

RPMI1

0.3

0.7

3.7

9.6

RPMI2

0.9

1.0

5.1

8.9

RPMI3

0.9

5.3

3.9

11.6

RPMI Mean Viability

 

99

99

RPMI Drift

 

-21%

-25%

LA Drift

 

-11%

12%

* Red values are either below 70% viability, above 150 S.I

Interpretation of results:
other: negative
Conclusions:
The test item showed no toxicity (No CV70 value) and no biologically relevant induction of the CD86 activity (No EC150 value) was measured at any of the test concentrations in both experiments. The test item is classified as negative in the U-Sens™ assay since negative results (< 150% increase) were observed at all test concentrations with a cell viability of >70% compared to the vehicle control.
In conclusion, the test item is classified as negative (no increase the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In chemico/in vitro skin sensitisation

There are three studies available, in which the substance was tested in chemico and in vitro for potential induction of skin sensitisation. All three studies were conducted under GLP conditons and in accordance with OECD Guidelines 442C (DPRA), 442D (KeratinoSens) and 442E (U-SENS), respectively.

-         Direct Peptide Reactivity Assay (DPRA)

The objective of this study was to determine the reactivity of the test item towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers following OECD Guidelines 442C.

In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed 27.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 13.5% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

-         KeratinoSens™ Assay

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay. Cells were incubated with the test item in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

The test item showed no toxicity (no IC30and IC50value) and no biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.25-fold and 1.15-fold in experiment 1 and 2 respectively. TA-1 is classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

In conclusion, the test item is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions of the study.

-         U-SENS

The objective of this study is to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay. The cell viability before incubation with the test item was > 90% (98% and 99% in experiment 1 and 2, respectively). Cells were incubated with the test item in a concentration range of 1.0 – 200 μg/mL and 20 – 200 μg/mL in experiment 1 and 2, respectively. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

The test item showed no toxicity (No CV70value) and no biologically relevant induction of the CD86 activity (No EC150 value) was measured at any of the test concentrations in both experiments. The test item is classified as negative in the U-Sens™ assay since negative results (< 150% increase) were observed at all test concentrations with a cell viability of >70% compared to the vehicle control.

In conclusion, the test item is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions of the study.

 

Based on the weight of evidence from the three available in chemico/in vitro studies, the substance is not considered to be skin sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data indicate that the substance does not meet the classification criteria for skin sensitisation in accordance with Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).


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