Registration Dossier

Administrative data

Description of key information

Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA, OECD TG 442C) but does activate keratinocytes (LuSens, OECD TG 422D). The third in vitro test (OECD TG 422E, h-CLAT) could not be performed because the substance is insoluble in all solvents indicated by OECD TG 442E at the required concentrations. Therefore, a Local Lymph Node Assay in mice was performed and determined the test item does not cause skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
29 October 2018 - 15 November 2018
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:
04. Feb. 2015
Deviations:
yes
Remarks:
see "Part Results"
Qualifier:
according to
Guideline:
other: EURL ECVAM (European Union Reference Laboratory for alternatives to animal testing): “DB-ALM Protocol n° 154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing.”
Version / remarks:
29. Jun. 2015
Deviations:
yes
Remarks:
see "Part Results"
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Synthetic peptides:
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, are used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

Instruments and Devices:
Components: Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A

Heating chamber, Centrifuge, Fridge, Freezer, Repeater pipette, pH-meter, Analytical scale, Analytical scale, Pipette 100 – 1000 µL, Glass thermometer, Ultrasonic bath, Conductometer, Carbon analyser

Chemicals:
Water for chromatography
H2O, Honeywell, HPLC grade
Demineralised water
H2O, from ion exchange cartridge. Total organic carbon (TOC) < 1 ppm
Acetonitrile for chromatography
CH3CN, ACN, Honeywell, HPLC grade
CH3CN, ACN, AppliChem, analysis grade
Trifluoroacetic acid
TFA, Merck, for spectroscopy
Hydrochloric acid
HCl, 1 M, p.a.
Ammonium hydroxide
NH3,25 %, p.a.
Ammonium acetate,
CH3COONH4, p.a, Sigma Aldrich
Disodium hydrogen phosphate,
Na2HPO4 * 2 H2O, p.a., Carl Roth

Buffers:
25 mM Phosphate buffer
1.1105 g disodium hydrogen phosphate dihydrate were dissolved in demineralised water, pH was adjusted to 7.5 with 1 M HCl (final volume 250 mL).
25 mM Ammonium acetate
481.9 mg ammonium acetate (anhydrous) were dissolved in demineralised water, pH was adjusted to 10.2 with 25 % ammonium hydroxide (final volume 250 mL).

Positive control:
Cinnamaldehyde (CAS 104-55-2, food grade ≥ 95 %),100 mM solution in acetonitrile for the cysteine peptide
2,3-Butanedione (CAS 431-03-8, > 97 %), 100 mM solution in acetonitrile for the lysine peptide
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion.

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.

Peptide stock solutions
The peptide stock solutions were freshly prepared for each assay.
0.667 mM Cys-Peptide solution was prepared by dissolving 22.5 mg of the peptide in 45 mL phosphate buffer, pH 7.5.
0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45 mL ammonium acetate buffer, pH 10.2.
0.667 mM Lys-Peptide solution was prepared by dissolving 18.1 mg of the peptide in 35 mL ammonium acetate buffer, pH 10.2. (experiment 2)
0.667 mM Lys-Peptide solution was prepared by dissolving 20.7 mg of the peptide in 40 mL ammonium acetate buffer, pH 10.2. (experiment 3)

Test item stock solution:
The test item stock solution is freshly prepared for each assay. 100 mM test item solution is prepared by dissolving 83.7 mg test item in 3 mL of the solvent acetonitrile for the Cys-Peptide and 83.7 mg test item for the Lys-peptide in experiment 1, respectively.
In experiment 2 and 3 the test item solution was prepared with 83.5 mg test item for the Lys-peptide measurement.

Peptide calibration standards:
From each peptide stock solution the following calibration standards were prepared in the appropriate dilution buffer: 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM peptide. Blank dilution buffer was also measured. Calibration samples were analysed before the samples containing the test item.

Test item samples:
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions described. A final volume of 1 mL per sample was prepared for each sample.

Incubation:
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 °C for 22 h and 25 min for the Cys-peptide and 22 h and 15 min for the Lys-peptide in experiment 1, respectively. In experiments 2 and 3 the incubation time was 22 h for the Lys-peptide. None of the Cys-peptide replicates were turbid after incubation in experiment 1. All three replicates for the Lys-peptide were turbid after incubation in experiment 1. They were centrifuged (10 min, 400 g) and only the clear supernatant was used for the measurement. In Experiment 2 and 3, none of the Lys-peptide replicates were turbid after incubation.

Measurements
HPLC system with UV/VIS-Detector

Evaluation of results:
Evaluation criteria of results according to the 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
0- ≤ 6.385 minimal or no reactivity negative

Evaluation criteria of results according to the cysteine 1:10 prediction model.
mean Cys peptide depletion [%] Reactivity Evaluation
> 98.24 - ≤ 100 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 13.89 ≤ 23.09 low reactivity positive
0 - ≤ 13.89 minimal or no reactivity negative

Acceptance criteria
The standard calibration curve should have an r² > 0.99
The mean peptide concentration of solvent control samples of sets A and C should be 0.50 ± 0.05 mM
The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100.0 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
The mean peptide depletion value for the positive control 2,3-butanedione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
The standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion.
Positive control results:
see "Any other information on results incl. tables"
Key result
Parameter:
other: mean peptide depletion for Cys-Peptide (%)
Run / experiment:
1st experiment
Value:
1.39
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion for Lys-Peptide (%)
Run / experiment:
1st experiment
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion for Lys-Peptide (%)
Run / experiment:
2nd experiment
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion for Lys-Peptide (%)
Run / experiment:
3rd experiment
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion for both peptides (%)
Value:
0.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The ten proficiency chemicals listed in the guideline were tested using the analysis method described. All ten proficiency chemicals showed the expected DPRA prediction and eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Calculated peptide depletion values for the Cys-Peptide

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

89.40

89.74

0.30

Positive control Rep. 2

89.84

Positive control Rep. 3

89.99

Test item Rep. 1

0.54

1.39

1.05

Test item Rep. 2

1.07

Test item Rep. 3

2.57

Table 2: Calculated peptide depletion values for the Lys-Peptide in experiment 1

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

44.02

46.48

2.49

Positive control Rep. 2

46.43

Positive control Rep. 3

49.00

Test item Rep. 1

0 (-0.7)*

0.00

0.00

Test item Rep. 2

0 (-0.5)*

Test item Rep. 3

0 (-1.08)*

* Note: Negative depletion values were considered as “zero” when calculating the mean.

Table 3: Calculated peptide depletion values for the Lys-Peptide in experiment 2

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

47.06

48.61

1.50

Positive control Rep. 2

48.71

Positive control Rep. 3

50.07

Test item Rep. 1

0 (-0.08)*

0.00

0.00

Test item Rep. 2

0 (-0.03)*

Test item Rep. 3

0 (-0.52)*

* Note: Negative depletion values were considered as “zero” when calculating the mean.

Table 4: Calculated peptide depletion values for the Lys-Peptide in experiment 3

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

44.72

47.16

2.49

Positive control Rep. 2

47.04

Positive control Rep. 3

49.71

Test item Rep. 1

0 (-0.71)*

0.00

0.00

Test item Rep. 2

0 (-0.46)*

Test item Rep. 3

0 (-0.93)*

Table 5: Historical Control Data for the positive control item

Parameter

Depletion [%]

Depletion [%]

Peptide

Cys- Peptide

Lys- Peptide

Mean

81.74

32.77

Standard
Deviation

8.48

3.99

Range 2σ

64.82 – 98.71

24.79 - 40.74

Conducted study

89.74

46.48 and 48.61 and 47.16

The following deviations of the guideline were observed:

The phosphate buffer used for dissolution of the Cys-peptide and the ammonium acetate buffer used for dissolution of the Lys-peptide in the experiments 1-3 were 25 mM instead of 100 mM. This was considered uncritical because the positive control confirmed that the buffer strength was sufficient.

The phosphate buffer used for dissolution of the Cys-peptide in experiment 1 was prepared by dissolving disodium hydrogen phosphate dihydrate and adjusting the pH by using 1 M HCl instead of using sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate in combination.This was considered uncritical because by dissolving disodium hydrogen phosphate in water, sodium dihydrogen ions are also formed and the pH was adjusted to the correct value.

The mean percent peptide-depletion of the positive control 2,3-butanedione was marginal out of range (the value was slightly too high) in the experiments 1, 2 and 3. This was considered uncritical, because it shows, that the test system was slightly too sensitive and even under these conditions the mean percent peptide-depletion of the test item showed a value for a clearly negative DPRA-prediction.

Table 6: Mean Peptide Depletion of both peptides

 

Cys-peptide
depletion [%]

Lys-peptide
depletion [%]

Mean peptide
depletion [%]

Experiment 1

1.39

0.00

0.70

Experiment 2

-*

0.00

Experiment 3

-*

0.00

*Note: in experiment 2 and 3, Cys-peptide was not performed, because a valid result was obtained in experiment 1.

Interpretation of results:
other: no peptide depletion
Conclusions:
The DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item possesses no or minimal skin sensitisation potential.
Executive summary:

A study according OECD TG 442C was performed in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile and the respective peptide was incubated at 25 °C for 22 h and 25 min for the Cys-peptide and 22 h and 15 min for the Lys-peptide, in experiment 1 respectively. In experiments 2 and 3 the incubation time was 22 h for the Lys-peptide. The peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

 

Experiment 1 was valid for the Cys-peptide. Within the measurement of the Lys-peptide the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

In experiment 2 only the measurement of Lys-peptide was performed and the mean peptide-depletion of the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

The third experiment was performed only for the Lys-peptide again and the mean peptide-depletion of the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

In experiments 1, 2 and 3 the mean peptide-depletion of the positive control 2,3-butanedione was only marginal too high, what shows that the system was a little too sensitive.

Due to the fact, that the mean peptide-depletion of the test item shows values for a clearly negative DPRA-prediction, these experiments were considered as valid. The result from experiment 1 was confirmed by the experiments 2 and 3.

 

In conclusion, the DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item possesses no or minimal skin sensitisation potential.

 

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 June 2018 - 28 June 2018
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:
Revised TG 442D – Draft v. 21. Dec. 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS. ENV/JM/MONO(2015)6.
Version / remarks:
22. May 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735:“In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”
Version / remarks:
14. Feb. 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: ESAC Opinion on the BASF coordinated Performance Standards based validation of the LuSens test method for skin sensitisation testing
Version / remarks:
No. 2016-04 of 24 June 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: BASF SE: Protocol LuSens Assay
Version / remarks:
16. May 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Preparation:
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and medium (Dulbecco´s Modified Eagle Medium, DMEM)). The test item was insoluble in DMEM but soluble in DMSO at the required concentration (200 mM) after shaking.
Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mM (CRFT) and 25 mM (experiment II and III) test item in DMSO was prepared and shaken. Subsequent dilution to 1 % finally yielded a maximum concentration of 2000 µM in the pre-test and 250 µM in the experiments. For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiments: factor 1.2) on a master plate. Afterwards all concentra-tions were further diluted (1:25) in medium no. 3 on a dilution plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the dilution plate to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

Controls:
Negative Control: DL-Lactic acid
Positive Control: EGDMA (Ethylene glycol dimethylacrylate)
Solvent Control: DMSO

Cell Cultures:
The LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany). For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of the laboratory to allow a continuous stock of cells (mycoplasma contamination free), which guarantees similar parameters of the experiment and reproducible characteristics of the cells. After thawing the cells were cultivated in DMEM (9 % FCS (Fetal calf serum) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Cytotoxicity Range Finder Test:
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the main experiments. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. A reduction of the viability below 70 % is defined as a cytotoxic effect. In the CRFT the following 12 nominal concentrations of the test item were tested: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1000 µM, 2000 µM

Experimental Parameters of Experiments:
In accordance with the OECD guideline 442D (draft version 21. Dec. 2017), the maximum final test item concentration should be 2000 µM. For a test chemical which has no defined molecular weight, the final test item concentration 2000 µg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility). In the case of a cytotoxic result, the concentrations for experiment II and III should be determined so that at least one of them is in the cytotoxic range.
Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations were chosen for experiment II and III:
33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM, 208.3 µM, 250.0 µM

Experimental Performance
Experiment II and III were performed in the same way. Experiment III serves only to confirm the results of experiment II.
At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83 000 (± 10 %) cells per mL. 120 µL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 25 h in experiment II and 23 h 15 min in experiment III.
The treatment procedure was performed on both 96 well plates identically: After the incubation time the medium was removed from the cells and 150 µL medium no. 3 were added to each well. Afterwards 50 µL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µL MTT working solution were added to each well. The plate was incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 100 µL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow colour) to its insoluble formazan (purple colour) in living cells and therefore indicates the amount of living cells. After the measurement of the colour change, the values were transferred in a validated spreadsheet for the calculation of the viability.
For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Afterwards 100 µL per well of a lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 µL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 µL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.




Positive control results:
The positive control induced a clear effect with an induction value of 4.8 (experiment II) and 4.9 (experiment III) fold in comparison to the solvent control.
Key result
Parameter:
other: fold Luciferase induction
Remarks:
100.5 µM
Run / experiment:
experiment II
Value:
1.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
120.6 µM
Run / experiment:
experiment II
Value:
1.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
144.7 µM
Run / experiment:
experiment II
Value:
1.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
173.6 µM
Run / experiment:
experiment III
Value:
1.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
208.3 µM
Run / experiment:
experiment III
Value:
1.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
250.0 µM
Run / experiment:
experiment III
Value:
2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use, the validity of the LuSens test at the laboratory was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LU-CIFERASE TEST METHODS) were tested. As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Results of experiment II

 

 

Induction of Luciferase

Viability of the Cells

Parameter

Concentration

Induction

Standard

Deviation

Standard

Deviation

Relative

Viability

Standard

Deviation

Standard

Deviation

 

[µM]

fold

 

[%]

[%]

 

[%]

Solvent Control

-

1.0

0.07

6.78

100.0

2.69

2.69

Growth Control

-

1.3

0.12

9.33

134.8

3.28

2.43

Negative Control

5000

1.0

0.03

2.75

100.9

2.60

2.58

Positive Control

120

4.8

0.36

7.52

81.3

2.99

3.68

Test item

33.6

1.4

0.05

3.64

91.7

1.34

1.46

Test item

40.4

1.5

0.15

10.12

94.2

2.34

2.49

Test item

48.5

1.4

0.15

10.25

91.8

2.80

3.05

Test item

58.1

1.4

0.03

1.96

88.4

1.76

1.99

Test item

69.8

1.6

0.04

2.55

89.2

1.77

1.99

Test item

83.7

1.6

0.06

3.75

88.1

2.14

2.43

Test item

100.5

1.9

0.12

6.42

81.0

1.50

1.85

Test item

120.6

1.9

0.13

7.02

80.4

2.29

2.85

Test item

144.7

1.9

0.08

4.29

72.5

2.21

3.05

Test item

173.6

2.0

0.10

5.30

68.3*

1.15

1.68

Test item

208.3

2.1

0.14

6.71

61.6*

2.25

3.65

Test item

250.0

2.4

0,17

7.05

55,0*

1.35

2.46

* = Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.

Table 2: Results of experiment II

 

 

Induction of Luciferase

Viability of the Cells

Parameter

Concentration

Induction

Standard

Deviation

Standard

Deviation

Relative

Viability

Standard

Deviation

Standard

Deviation

 

[µM]

fold

 

[%]

[%]

 

[%]

Solvent Control

-

1.0

0.07

6.82

100.0

6.69

6.69

Growth Control

-

1.1

0.09

7.97

153.5

7.60

4.95

Negative Control

5000

1.0

0.05

4.75

106.7

2.67

2.50

Positive Control

120

4.9

0.35

7.02

88.9

5.08

5.72

Test item

33.6

1.2

0.06

5.46

109.2

3.19

2.92

Test item

40.4

1.2

0.06

4.85

111.5

8.21

7.37

Test item

48.5

1.2

0.07

5.79

113.4

1.21

1.06

Test item

58.1

1.3

0.02

1.50

106.0

7.60

7.16

Test item

69.8

1.3

0.07

5.33

102.5

8.57

8.36

Test item

83.7

1.4

0.11

8.03

100.2

8.93

8.91

Test item

100.5

1.5

0.13

8.26

102.5

4.03

3.93

Test item

120.6

1.6

0.16

9.69

100.8

2.34

2.32

Test item

144.7

1.7

0.01

0.83

96.0

6.40

6.67

Test item

173.6

1.8

0.23

12.71

85.4

1.41

1.65

Test item

208.3

1.8

0.33

18.21

79.9

5.23

6.55

Test item

250.0

2.0

0.04

2.06

74.0

1.13

1.53

Table 3: Acceptability of experiment II and III

Criteria

Found in

experiment II

Found in

experiment III

The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.

Positive controlFold induction:

4.8

Relative viability: 81.3 %

Positive controlFold induction:

4.9

Relative viability:  88.9 %

The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.

Negative control:

Fold induction:

1.0

Relative viability:

100.9 %

Growth control:

Fold induction:

1.3

Relative viability:

134.8 %

Negative control:

Fold induction:

1.0

Relative viability:

106.7 %

Growth control:

Fold induction:

1.1

Relative viability:

153.5 %

The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.

6.78 %

6.82 %

At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.

9 concentrations are analysable

12 concentrations are analysable

Table 4: Historical Data of the Negative Control and the Positive Control (status: 18. May 2018)

 

Σ

Mean induction value

Standard

deviation

Range

Study No.:18042602G887

 

 

 

 

 

Experiment II

Experiment III

Positive Control

(EGDMA)

152

5.5

1.566

2.1 – 14.7

4.8

4.9

Negative control

(DL-Lactic acid)

172

1.0

0.080

0.8 – 1.3

1.0

1.0

Interpretation of results:
other: activation of keratinocytes
Conclusions:
Under the experimental conditions of this study, the test item was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).
Executive summary:

An in vitro study according OECD TG 442D was performed to investigate the potential of the test item to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

In total three experiments were performed of which one was invalid (experiment I) and had to be repeated. In the end two valid experiments were performed and evaluated. Therefore two independent experiments were performed. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment II and III:

33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM, 208.3 µM, 250.0 µM

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control. The induction of the luciferase of the growth control was < 1.5 fold. Since all acceptability criteria of the assay were met the study is valid.

In experiment II cytotoxic effects were observed in the following concentrations:

173.6 µM, 208.3 µM, 250.0 µM

Those concentrations were excluded from the evaluation of the luciferase induction.

In experiment III no cytotoxic effects were observed.

A statistically significant increase ≥ 1.5 fold in luciferase induction was measured in the following concentrations in experiment II:

40.4 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM

A statistically significant increase ≥ 1.5 fold in luciferase induction was measured in the following concentrations in experiment III:

100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM, 208.3 µM, 250.0 µM

Therefore, both experiments are clearly positive.

In conclusion, it can be stated that under the experimental conditions of this study, the test item was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 July 2019 to 30 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/Ca Ola Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: DRF: 9-10 weeks; Main study: 10 weeks
- Weight at study initiation: 18.9 – 21.5 g (main study)
- Housing: Acclimation: grouped caging in small groups; Study: grouped caging (4 animals/ Type II. polypropylene/polycarbonate cage)
- Diet: ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 7 days
- Indication of any skin lesions: not showing any sign of skin lesion

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: Absolute ethanol: water 7:3 (v/v) (EtOH)
Concentration:
5, 10, 25 and 50 %
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The best solubility was observed in Absolute ethanol: water 7:3 (v/v) with a maximum concentration of 50 % (w/v). Further vehicles recommended in the relevant guidelines were evaluted but only lower maximum soluble concentrations were achieved (25 % or 10 %, w/v). Therefore, Absolute ethanol: water 7:3 (v/v) was selected as vehicle. For the dose range finder (DRF) 50 and 25 % concentrations were used.
- Irritation: No sign of significant irritation (indicated by an erythema score ≥ 3 and/or an increase of ≥ 25 % of ear thickness observed on any day of measurement) or any other local effect was observed.
- Systemic toxicity: No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed.
- Ear thickness measurements: The % deviation on day 3 compared to pre-treatment was max. 10 % for 50 % concentration and max. 15 % for 25 % concentration. On day 6 no deviation from the pre-treatement value was detected.
- Erythema scores: Scores were 0 for both animals on treatment days (days 1-3) and afterwards (days 4-6).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
The animals were randomly assigned to control and test groups using a randomization scheme.
- Name of test method: Local Lymph Node Assay (Pooled Treatment Group Approach)
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer if exposure to at least one concentration (non-irritating, non-toxic) of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). Furthermore, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses were considered when determining whether a borderline result was declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was weighed and the homogeneous formulations in the selected vehicle of Absolute ethanol: water 7:3 (v/v) mixture were prepared with the vehicle in a final volume of 1 mL using volumetric vials and mechanical agitation. All formulations were freshly prepared just before the daily treatments.
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There were no treatments on Days 4, 5 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All calculations were made by Microsoft Excel Software.
Positive control results:
The positive control group animals were treated with 25 % HCA solution (formulated in AOO) concurrent to the test item treated groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. The positive control substance induced the appropriate stimulation compared to the relevant control (AOO). The calculated SI value was 7.9. The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
test item concentration of 50 %
Parameter:
SI
Value:
2.1
Test group / Remarks:
test item concentration of 25 %
Parameter:
SI
Value:
1.1
Test group / Remarks:
test item concentration of 10 %
Parameter:
SI
Value:
1.6
Test group / Remarks:
test item concentration of 5 %
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. Appearance of the lymph nodes was normal in the negative control groups (AOO or EtOH) and in the test item treated groups.

DETAILS ON STIMULATION INDEX CALCULATION
No significant lymphoproliferative response (SI ≥ 3) was observed for the test item at the tested concentrations. The observed stimulation index values were 1.1, 2.1, 1.1 and 1.6 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using all SI values obtained. No significant dose-response correlation was observed (p = 0.78, r2 = 0.05).

EC3 CALCULATION
Based on the SI values no EC3 was calculated.

CLINICAL OBSERVATIONS
No mortality or symptoms of systemic toxicity were observed during the test. No erythema or other local effect was observed in any test group.

BODY WEIGHTS
Body weight decrease by > 5 % was observed in the AOO group only (1 of the 4 animals, 6 % decrease). No body weight decrease by > 5 % was observed in the other test groups.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was determined not to cause skin sensitisation in an LLNA study in mice.
Executive summary:

A Local Lymph Node Assay in mice according to OECD 429 was performed using the test substance. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and a Dose Range Finding test (DRF).

Solubility of the test item was evaluated in suitable vehicles. The best solubility was observed in Absolute ethanol: water 7:3 (v/v) mixture (EtOH) with a maximum concentration of 50 % (w/v).

In the DRF no adverse effects (systemic toxicity or irritation) were observed up to this maximum concentration. According to this the test item was examined in the LLNA at 50 %, 25 %, 10 % or 5 % (w/v) concentrations as formulations in EtOH.

Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and negative control groups dosed with the vehicle of the positive control or the test item (AOO or EtOH, respectively) were employed.

The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the control (SI = 7.9). Thus, confirming the sensitivity and validity of the assay.

No mortality was observed. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any test group. No signs of irritation (monitored by erythema scoring) or any other local effects were observed at the treatment site (ears) in any test group.

No significant lymphoproliferative response (SI ≥ 3) was observed for the test item at the tested concentration. The observed stimulation index values were 1.1, 2.1, 1.1 and 1.6 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. No significant dose-related response was observed (evaluated by linear regression using the SI values; p =0.78, r =0.05).

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 50 % (w/v, based on solubility) as well as the lack of a significant dose-related response is considered as evidence that the test substance is not a skin sensitizer.

Under the conditions of the present assay, the test substance formulated in a suitable vehicle (Absolute ethanol: water 7:3 (v/v) mixture, EtOH) and tested at the maximum concentration of 50 % (w/v, based on solubility) and also at 25 %, 10 % or 5 % (w/v) concentrations was shown to have no skin sensitization potential in the Local Lymph Node Assay in mice.

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

For the evaluation of the skin sensitisation potential of the test substance a weight of evidence approach was used. Therefore, an in chemico/ in vitro test strategy was employed first. As no clear result was reached a Local Lymph Node Assay was performed.

 

Studies

DPRA

A study according OECD TG 442C was performed in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile and the respective peptide was incubated at 25 °C for 22 h and 25 min for the Cys-peptide and 22 h and 15 min for the Lys-peptide, in experiment 1 respectively. In experiments 2 and 3 the incubation time was 22 h for the Lys-peptide. The peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Experiment 1 was valid for the Cys-peptide. Within the measurement of the Lys-peptide the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

In experiment 2 only the measurement of Lys-peptide was performed and the mean peptide-depletion of the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

The third experiment was performed only for the Lys-peptide again and the mean peptide-depletion of the positive control 2,3-butanedione was marginal out of the range (the value was slightly too high).

In experiments 1, 2 and 3 the mean peptide-depletion of the positive control 2,3-butanedione was only marginal too high, what shows that the system was a little too sensitive.

Due to the fact, that the mean peptide-depletion of the test item shows values for a clearly negative DPRA-prediction, these experiments were considered as valid. The result from experiment 1 was confirmed by the experiments 2 and 3. The DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item possesses no or minimal skin sensitisation potential.

 

LuSens

An in vitro study according OECD TG 442D was performed to investigate the potential of the test item to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

In total three experiments were performed of which one was invalid (experiment I) and had to be repeated. In the end two valid experiments were performed and evaluated. Therefore, two independent experiments were performed. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment II and III:

33.6 µM, 40.4 µM, 48.5 µM, 58.1 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM, 208.3 µM, 250.0 µM

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control. The induction of the luciferase of the growth control was < 1.5 fold. Since all acceptability criteria of the assay were met the study is valid.

In experiment II cytotoxic effects were observed in the following concentrations:

173.6 µM, 208.3 µM, 250.0 µM

Those concentrations were excluded from the evaluation of the luciferase induction.

In experiment III no cytotoxic effects were observed.

A statistically significant increase ≥ 1.5 fold in luciferase induction was measured in the following concentrations in experiment II:

40.4 µM, 69.8 µM, 83.7 µM, 100.5 µM, 120.6 µM, 144.7 µM

A statistically significant increase ≥ 1.5 fold in luciferase induction was measured in the following concentrations in experiment III:

100.5 µM, 120.6 µM, 144.7 µM, 173.6 µM, 208.3 µM, 250.0 µM

Therefore, both experiments are clearly positive.

It can be stated that under the experimental conditions of this study, the test item was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

 

h-CLAT

The in vitro study according to OECD 422E could not be performed as the test item was not soluble at the required concentrations in the solvents indicated in the guideline.

 

LLNA

A Local Lymph Node Assay in mice according to OECD 429 was performed using the test item. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and a Dose Range Finding test (DRF).

Solubility of the test item was evaluated in suitable vehicles. The best solubility was observed in Absolute ethanol: water 7:3 (v/v) mixture (EtOH) with a maximum concentration of 50 % (w/v).

In the DRF no adverse effects (systemic toxicity or irritation) were observed up to this maximum concentration. According to this the test item was examined in the LLNA at 50 %, 25 %, 10 % or 5 % (w/v) concentrations as formulations in EtOH.

Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and negative control groups dosed with the vehicle of the positive control or the test item (AOO or EtOH, respectively) were employed.

The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the control (SI = 7.9). Thus, confirming the sensitivity and validity of the assay.

No mortality was observed. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any test group. No signs of irritation (monitored by erythema scoring) or any other local effects were observed at the treatment site (ears) in any test group.

No significant lymphoproliferative response (SI ≥ 3) was observed for the test item at the tested concentration. The observed stimulation index values were 1.1, 2.1, 1.1 and 1.6 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. No significant dose-related response was observed (evaluated by linear regression using the SI values; p =0.78, r =0.05).

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 50 % (w/v, based on solubility) as well as the lack of a significant dose-related response is considered as evidence that the test item is not a skin sensitizer.

Under the conditions of the present assay, the test item formulated in a suitable vehicle (Absolute ethanol: water 7:3 (v/v) mixture, EtOH) and tested at the maximum concentration of 50 % (w/v, based on solubility) and also at 25 %, 10 % or 5 % (w/v) concentrations was shown to have no skin sensitization potential in the Local Lymph Node Assay in mice.

 

Conclusion:

Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA, OECD TG 442C) but does activate keratinocytes (LuSens, OECD TG 422D). The third in vitro test (OECD TG 422E, h-CLAT) could not be performed because the substance is insoluble in all solvents indicated by OECD TG 442E at the required concentrations. As no final conclusion on the skin sensitizing potential of the test item was possible with the in vitro/in chemico test strategy a Local Lymph Node Assay (LLNA, OECD 429) in mice was conducted. The LLNA showed that the test item does not cause skin sensitization in vivo. Therefore, the test item is determined to have no skin sensitising potential using the weight of evidence approach.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on the available experimental test data, the test substance is not considered to have skin sensitisation potential under Regulation (EC) No 1272/2008, as amended for the 12th time in Regulation (EU) 2019/521.