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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-03-23 to 2015-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
skin sensitisation: in chemico
Remarks:
in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-03-16 to 2015-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Test material information:
Composition 1
Details on study design:
TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: acetonitrile
- Reason for the vehicle: The test substance is soluble in acetonitrile.

CONTROLS
- Negative control: vehicle control: acetonitrile
- Concurrent control: vehicle control with the peptides
- Co-elution control: buffer and test substance without the peptide
- Positive control: ethylene glycol dimethacylate (50 nM solution in acetonitrile)

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
-- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol).
- Stock solution:
-- C-containing peptide: 0.667 mM of peptide in pH 7.5 phosphate buffer
-- K-containing peptide: 0.667 mM of peptide in pH 10.2 ammonium acetate buffer
- Ratios
- C-containing peptide: ratio of 1:10 (0.5 mM peptide, 5mM test substance)
- K-containing peptide: ratio of 1:50 (0.5 mL peptide, 25 mM test substance)

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220 nm: HPLC analysis started 24 hours after sample preparation and the analysis time was 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours and visually investigated for any precipitate that may occur during the exposure period.
- Preparation of vehicle control: prepared in triplicates in the same way as the test substance samples described above but with acetonitrile instead of the test subtance.
- Preparation of co-elution control: prepared in the same way as the test substance sampled described above but without the peptide, instead buffer was used.

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Agilent PH 1100
- Wavelength: 220 nm and 258 nm
- Detection: Diode Array Detector

DATA EVALUATION
- Calculation of the peptide concentrations: peptide concentration (mM) = [peak area (mAU * s) - b] / m with b: axis intercept and m: slope
- Calculation of the peptide depletion: peptide depletion of sample = [ 1 - (peptide concentration of sample (mM) / mean peptide concentration of NC (mM))] * 100%
- Mean peptide depletion for each peptide = C - containing peptide depletion of a test substance (%) = mean (C - containing peptide depletion of samples 1-3) (%)
- Mean peptide depletion = (C - containing peptide depletion + K - containing peptide depletion) / 2

ACCEPTANCE CRITERIA
- The positive control causes depletion of both peptides comparable to historic data.
- The standard calibration curve should have an r2 > 0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of nine vehicle controls B and C should be < 15%
- The variability should be acceptable low (SD < 14.9% for cysteine depletion and < 11.6% for lysine depletion)

EVALUATION RESULTS
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion >42.47
-- moderate: mean peptide depletion >22.62 =42.47
-- low: mean peptide depletion >6.38 =22.62
-- minimal: mean peptide depletion =6.38
High, moderate and low reactivity are evaluated as positive, while minimal reactivity is evaluated as negative.
- In case the mean peptide depletion cannot be determined due to invalid K-peptide depletion the evaluation is performed as follows:
-- high: mean peptide depletion >98.24
-- moderate: mean peptide depletion >23.09 =98.24
-- low: mean peptide depletion >13.89 =23.09
-- minimal: mean peptide depletion =13.89
High, moderate and low reactivity are evaluated as positive, while minimal reactivity is evaluated as negative.
Positive control results:
Positive controls depleted cysteine and lysine peptides by 55.05 % (SD 5.56) and 14.5 % (SD 1.48) respectively. The mean peptide depletion of the positive control was 34.77%.
Key result
Parameter:
other: Peptide depletion
Run / experiment:
C-containing peptide (mM)
Value:
-0.52
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Peptide depletion
Run / experiment:
K-containing peptide (mM)
Value:
-0.04
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean peptide depletion
Value:
0
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
Solubility: The test substance was soluble in acetonitrile. The samples of the test substance with the Cpeptide were solutions immediately after preparation and after 24 hours. The samples with the K-peptide were homogeneous emulsions immediately after preparation and after 24 hours.

ACCEPTANCE OF RESULTS:
A study is considered acceptable if the positive control causes depletion of both peptides
comparable to historic data.
The standard calibration curve should have an r² >0.99.
The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05
mM.
The CV of the nine vehicle controls B and C should be < 15%.
Since the mean peptide depletion for each peptide is determined from the mean of three single
samples, the variability between these samples should be acceptably low (SD <14.9% for %
cysteine depletion and <11.6% for % lysine depletion).

1. Results Cysteine-containing peptide:

DPRA: Peak area, peptide concentration and peptide depletion of NC, PC and test substance for cysteine-peptide

Reaction with cysteine-peptide Peak area (mAU*s) at 220 nm Peptide concentration (mM)
Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 Mean SD
NC: ACN 857.6 843.8 582.0 0.482 0.474 0.479 0.478 0.004
Test item 860.0 852.3 854.4 0.483 0.479 0.480 0.481 0.002
PC: EGDMA in ACN 431.6 380.4 337.1 0.242 0.214 0.189 0.215 0.027
Reaction with cysteine-peptide Peptide depletion (%)          
Sample 1 Sample 2 Sample 3 Mean SD      
NC: ACN -0.76 0.86 -0.10 0.00 0.82      
Test item -1.04 -0.13 -0.38 -0.52 0.47      
PC: EGDMA in ACN 49.34 55.36 60.46 55.05 5.56      

2. Results Lysine-containing peptide:

DPRA: Peak area, peptide concentratio and peptide depletion of NC, PC and test substance for lysine-peptide
Reaction with Lysine-peptide Peak area (mAU*s) at 220 nm Peptide concentration (mM)
Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3 Mean SD
NC: ACN 745.4 748.7 747.6 0.496 0.498 0.497 0.497 0.001
Test item 760.3 742.4 739.9 0.506 0.494 0.492 0.497 0.007
PC: EGDMA in ACN 650.0 639.2 627.8 0.432 0.425 0.417 0.425 0.007
Reaction with Lysine-peptide Peptide depletion (%)          
Sample 1 Sample 2 Sample 3 Mean SD      
NC: ACN 0.25 -0.20 -0.05 0.00 0.23      
Test item -1.74 0.65 0.99 -0.04 1.49      
PC: EGDMA in ACN 13.03 14.46 16.0 14.5 1.48      

3. Mean peptide depletion:

For the test substance the mean peptide depletion as average of cysteine and lysine-peptide depletions is calculated. Negative depletions were considered to be zero for calculation:

  Cysteine-peptide  Lysine-peptide Mean of both depletions (%)
  Mean depletion (%) SD Mean depletion (%) SD
Test substance -0.52 0.47 -0.04 1.49 0.00
PC: EGDMA in ACN 55.05 5.56 14.5 1.48 34.77

4. Evaluation criteria of DPRA:

Cysteine 1:10 / Lysine 1:50 prediction model

Mean peptide depletion (%) Reactivity Evaluation
>42.47 high reactivity positive
 >22.62 =42.47 moderate reactivity positive
> 6.38 =22.62 low reactivity positive
=6.38 minimal or no reactivity negative

5. Acceptance criteria:

The acceptance criteria were met with the exception of the vehicle control of the K-peptide samples which was not available due to a technical error. However, the test is considered to be valid, as all other control samples met the criteria.

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance shows no chemical reactivity in the DPRA test under the current conditions. Therefore, the test item is not considered not to be a sensitiser.
Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)- containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was assessed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

The test substance was soluble in acetonitrile. The samples of the test substance with the C-peptide were solutions immediately after preparation and after 24 hours. The samples with the K-peptide were homogeneous emulsions immediately after preparation and after 24 hours. The samples of both peptides were centrifuged prior to HPLC analysis. The mean C-peptide depletion, caused by the test substance was determined to be -0.52%. The mean K-peptide depletion, caused by the test substance was determined to be -0.04%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.0%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model it was concluded that the test item shows a minimal chemical reactivity in the DPRA under the test conditions chosen. In conclusion, the test item is not a sensitiser.

Reason / purpose:
reference to same study
Reference
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-04-2 to 2015-08-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD guideline 442E h-CLAT
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dentritic cells
Test material information:
Composition 1
Details on study design:
TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: American Type Culture Collection Manassas, USA (ATCC, TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentrations: 1st experiment: 2027, 1689, 1408, 1173, 977, 815, 679 and 566 µg/mL; 2nd experiment: 1173, 977, 815, 679, 566, 471, 393 and 327 µg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Vehicle: culture medium
- Reason for vehicle: The h-CLAT was performed in an aqueous test system.

CONTROLS
- Negative control (NC): Lactic acid (LA); 1000 µg/mL in culture medium
- Vehicle control (VC): Culture medium
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB); 4.0 µg/mL in 0.2% DMSO in culture medium

MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25 mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma A7030)
- Blocking Solution: 0.01% Globulins Cohn fraction II,III (Sigma G2388) with DPBS (without Ca2+ / Mg2+)
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 2
- Experiments: 2
- Exposure period: 24 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2 -mercaptethanol (30 > passage >= 5)

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 hours after exposure

DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (cell viability of test substance treated cells / mean cell viability of vehicle control treated cells) * 100
- Relative fluorescence intensity: RFI (%) = (MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells) * 100
- Calculation of EC 150% and EC 200%: If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment. The calculation is performed by linear regression from the two concentrations directly above and below the EC 150% / EC 200% concentration.

ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when viability is less than 50%
- Cell viability of vehicle control cells must yield at least 90%
- In the positive control (DNCB), RFI (relative fluorescence intensity)
values of both CD86 and CD54 should be exceed the positive criteria (RFI CD86 =150% and CD54 =200%) and cell viability should be =50%.
- For all vehicle control, the MFI (mean fluoresence intensity) ratio of both CD86 and CD54 to isotype controls should be =105%.
- A study is considered to be acceptable if the positive, negative and vehicle control data lies within the range of the historic data.

EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased =150% and/or CD54 expression increased =200% in relation to vehicle control in at least 2 independent experiments.
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 µg/mL for the vehicle culture medium or 2000 µg/mL for 0.2% DMSO in culture medium).
Positive control results:
Positive controls achieved RFI values of both CD86 and CD54 over the positive criteria (CD86= 311.9% and CD54 = 368.1%) and cell viability was 82.1%.
Key result
Parameter:
other: RFI CD54 mean (%) and RFI CD54 mean (%)
Run / experiment:
Please see 'Any other information on results incl. tables'.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation

Mean value and standard deviation for 1st experiment:

Concentration (test substance) µg/mL RFI CD86 RFI CD54 Rel. Viability (%) SD of viability
mean SD mean SD
566 169.9 20.3 320.5 6.8 89.0 2.8
679 180.6 29.0 288.5 62.3 75.4 1.3
815 160.8 13.2 395.5 16.5 67.6 4.8
977 150.9 0.8 333.3 22.8 14.6 1.4
1173 221.5 1.8 418.7 106.4 53.0 23.8
1408 198.4 28.8 582.5 72.4 63.7 9.8
1689 218.4 19.5 607.1 78.6 53.0 3.3
2027 78.0 114.5 506.4 38.1 39.3 2.4
VC 100.0 - 100.0 - 100.0 0.2
LA 1000 µg/mL 79.5 10.7 124.1 25.5 99.6 0.2
DNCB 4 µg/mL 311.9 88.5 368.1 70.2 82.1 3.7

Mean value and standard deviations fo 2nd experiment:

Concentration (test substance) µg/mL RFI CD86 RFI CD54 Rel. Viability (%) SD of viability
mean SD mean SD
327 102.1 4.6 240.7 15.3 100.4 0.3
393 96.1 0.1 236.4 41.8 100.3 0.3
471 106.3 3.4 382.3 47.8 100.0 0.1
566 100.2 1.3 364.9 189.4 99.7 0.1
679 107.2 4.7 422.1 66.3 98.3 0.8
815 127.0 9.8 557.8 89.0 78.5 3.9
977 130.0 12.2 737.3 65.9 71.0 0.9
1173 129.1 5.7 592.7 150.1 56.2 1.7
VC 100.0 - 100.0 - 100.0 100.0
LA 1000 µg/mL 66.9 1.4 104.9 14.2 100.3 0.2
DNCB 4 µg/mL 300.0 19.7 392.6 0.9 84.4 1.1
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the results and the prediction model, it is concluded that the test substance induces dendritic cell activation (CD54 expression increased =200% in at least one assay) and is therefore positive for skin sensitisation.

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

 

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 2 valid experiments were performed. In the main experiment the test substance was an emulsion in culture medium (2 x stock preparations) at concentrations of 566 µg/mL onwards. Lower concentrations were solved. In 0.2% DMSO in culture medium solutions were noticed at all concentrations (final concentrations). No precipitates were noticed in any concentration after 24 hours. In at least two independent experiments an induction of the expression of CD54 (above 200%) was observed at sufficiently non-cytotoxic (cell viability = 50%) concentration.

 

In summary, it has to be concluded that test substance induces dendritic cell activation and is therefore considered to be positive for skin sensitization.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on study design:
TEST SYSTEM
- Cell line: Human transgenic keratinocyte cell line derived from HaCaT cells
- Source: prepared in collaboration with Christoph J. Wruck, RWTH Aachen

TEST-SUBSTANCE PREPARATION
- Concentrations: 1941, 1618, 1348, 1123, 936, 780, 650 and 542 µg/mL
- Stock: 4x concentration of the highest concentration
- Vehicle: 4% DMSO in culture medium 3
- Reason for vehicle: 4% DMSO in culture medium 3 was used because good homogeneity of the preparation was achieved

CONTROLS
- Negative control: DL-Lactic acid (LA, CAS n° 50-21-5), 450 µg/mL in 1% DMSO in culture 3
- Concurrent control: Vehicle control (VC): 1% DMSO in culture medium 3; Blank control: Culture medium 3 without cells; Basal control: Culture medium 3 with cells
- Positive control: Ethylene glycol dimethacrylate (EGDMA, CAS no. 97-90-5), 18 µg/mL in 1% DMSO in culture medium 3

MEDIUM
- Culture medium 1: D-MEM (Cat. No. Biochrom FG 0445) + 10 % FBS + 1 % Penicillin / Streptomycin, Puromycin dihydrochloride 25 µL (Sigma P9620- 10mL)
- Culture medium 2: D-MEM (Cat. No. Biochrom FG 0445) + 10 % FBS
- Culture medium 3: D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS
- Buffer: Phosphate Buffered Saline (PBS) w/o Ca2+ / Mg2+ + 0.05 % EDTA Phosphate Buffered Saline (PBS) w/o Ca2+ / Mg2+ Phosphate Buffered Saline (PBS) with Ca2+ / Mg2+

EXPERIMENTAL PROCEDURE
-Replicates: 3
-Experiments: 3
-Exposure period: 48 hours
-MTT assay: in Medium 3
- Washing cells: PBS (with Ca2+/Mg2+)
- Incubation: 2 hours
-Luciferase assay: in Medium 3
- Washing cells: PBS (with Ca2+/Mg2+)
- Buffer: Steady-Glo-preparation (= 100 µL PBS (without Ca2+/Mg2+))
- Incubation: 10 min, room temperature, in darkness

ANALYSIS
- Absorbance measurement: at 570 nm with reference wavelength 690 nm

DATA EVALUATION
- CV 75 Calculation: relative survival rate was calculated by linear extrapolation. This value is the substance concentration at which relative cell viability is 75% compared to the vehicle control.
- % relative cell viability = (absorbance of test substance treated cells - absorbance of blank) / (absorbance of mean vehicle control treated cells - absorbance of blank) * 100
- Luciferase fold induction = (test substance treated cells - background without cells) / (mean vehicle control treated cells - background without cells)

ACCEPTANCE CRITERIA
- The cells viability of vehicle control must be at least 90%
- The mean of the positive control EGDMA should achieve =2.50 fold induction
- The mean of the LA (negative control) <1.50
- The mean of the viability must be =70%
- The cell viability (%) of luminescence in the vehicle control wells for each plate should be <20%
- The mean of the basal expression of the cells must be <1.5 as compared to the solvent control.
- The positive, negative and vehicle control data must lie within the range of the historic data.

EVALUATION RESULTS
A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least consecutive concentrations of two independent experiments.
A test substance is considered to be "negative" when the criteria mentioned above are not met up to the maximum concentration (=2000 µg/mL).

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: relative cell viability
Run / experiment:
Mean of 3 experiments
Value:
> 70
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: EC1.5
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Calculation of an EC1.50 was not applicable

Any other information on results incl. tables

1. Preliminary cytotoxicity assessment:

Results of preliminary cytotoxicity assessment:
Concentration (active ingredient) µg/mL Concentration (test substance) µg/mL mean OD 570-690 of 3 replicates Mean rel. Viability (%)
VC VC 0.392 100.0
0.5 0.5 0.335 85.5
1 1 0.326 83.1
5 5 0.353 90.0
10 10 0.359 91.7
50 50 0.327 83.3
100 100 0.319 81.4
500 501 0.328 83.8
1000 1001 0.321 82.0
2000 2002 0.098 24.9

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 1123µg/mL.

2. Experiment 2: Mean values and standard deviations of luciferase induction and rel. viability as well as p-value of t-test. Concentration with fold induction above 1.50 with rel. viability = 70% and with statistical significance are indicated in bold.

Concentration (test substance) µg/mL Fold induction Rel. Viability (%) t-test
mean SD mean SD p-value markers
542 1.22 0.10 90.9 5.3 0.024 *
650 1.15 0.21 97.9 6.2 0.176 n.s.
780 1.16 0.19 103.8 4.2 0.145 n.s.
936 1.06 0.12 97.3 9.4 0.252 n.s.
1123 1.11 0.13 91.7 8.6 0.142 n.s.
1348 1.04 0.04 96.4 8.7 0.107 n.s.
1618 1.12 0.06 88.0 7.5 0.022 *
1941 1.17 1.00 30.4 22.6 0.398 n.s.
VC 1.00 0.09 100.0 6.8 - -
EGDMA (18 µg/mL) 8.12 0.91 96.8 4.3 0.000 **
LA (450 µg/mL) 0.83 0.08 101.1 6.7 0.002 **

3. Experiment 3: Mean value and standard deviations of luciferase induction and rel. viability as well as p-values of t-test. Concentration with fold inductions above 1.50 with rel. viability = 70% and with statistical significance are indicated in bold.

Concentration (test substance) µg/mL Fold induction Rel. Viability (%) t-test
mean SD mean SD p-value markers
542 1.23 0.14 83.6 2.4 0.046 *
650 1.48 0.17 77.8 3.5 0.015 *
780 1.43 0.09 87.3 5.7 0.001 **
936 1.22 0.13 82.8 3.2 0.044 *
1123 1.47 0.13 81.4 4.6 0.008 **
1348 1.08 0.08 86.7 4.2 0.112 n.s.
1618 1.23 0.10 88.8 5.8 0.019 *
1941 1.46 0.19 78.4 3.2 0.022 *
VC 1.00 0.14 100.0 5.3 - -
EGDMA (18 µg/mL) 7.52 0.27 105.9 7.5 0.000 **
LA (450 µg/mL) 0.86 0.16 107.5 4.1 0.060 n.s.

4. Experiment 4: Mean value and standard deviations of luciferase induction and rel. viability as well as p-values of t-test. Concentration with fold inductions above 1.50 with rel. viability = 70% and with statistical significance are indicated in bold.

Concentration (test substance) µg/mL Fold induction Rel. Viability (%) t-test
mean SD mean SD p-value markers
542 1.12 0.12 88.5 8.5 0.109 n.s.
650 1.12 0.16 84.0 8.7 0.168 n.s.
780 1.22 0.25 92.4 1.7 0.134 n.s.
936 1.24 0.07 86.4 6.7 0.002 **
1123 1.22 0.23 93.0 5.4 0.118 n.s.
1348 1.22 0.07 86.8 6.4 0.002 **
1618 1.25 0.04 81.2 7.8 0.000 **
1941 1.22 0.10 45.0 13.9 0.017 *
VC 1.00 0.18 100.0 9.1 - -
EGDMA (18 µg/mL) 6.99 0.76 101.4 8.1 0.000 **
LA (450 µg/mL) 1.11 0.12 110.4 6.1 0.067 n.s.

Mean of 3 experiments

Concentration (test substance) µg/mL

Fold induction

Rel. Viability (%)

mean

SD

mean

SD

542

1.19

0.06

87.67

3.72

650

1.25

0.20

86.57

10.29

780

1.27

0.14

94.5

8.45

936

1.17

0.10

88.83

7.55

1123

1.27

0.18

88.7

6.36

1348

1.11

0.10

89.97

5.57

1618

1.20

0.07

86.00

4.18

1941

1.29

0.15

50.6

23.51

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item does not have keratinocyte activating potential. Therefore, the test item is not considered not to be a skin sensitiser.
Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

 

In the main test, luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. At concentrations used in the main experiment the test substance was an emulsion in 4% DMSO in culture medium 3 (4 x stock preparations) and a solution in 1% DMSO in culture medium 3 (final concentrations) at all concentrations. No precipitates were noticed in any concentration after 48 hours. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable.

 

In summary, after 48 hours of exposure to the test substance, luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential. Therefore, the test item is not considered a non-sensitiser.