Tribenzylamine

Endpoint summary

Description of key information

The test item is predicted by DPRA as negative and not to be a potential skin sensitizer. However, due to precipitation the negative result cannot be used in an assessment of skin sensitisation potential (reference 7.4.1 -1).

The test item activated LuSens cells under the test conditions of this study. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP) (reference 7.4.1-2).

The test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP) (reference 7.4.1-3).

Overall, the test item is predicted to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-12-01 to 2020-12-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

U937 cell line activation test (U-SENS™)

Details of test system:

U-937 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: On the day of the experiment, the test item was dissolved in DMSO to prepare a stock solution with a concentration of 50 mg/mL.
- Preparation of the test chemical serial dilutions: Dilutions in DMSO were prepared from the stock solution and further dilutes with culture medium. By mixing the dilutions with the cell suspension, the final treatment concentrations were achieved.
- Preparation of the positive control: in culture medium
- Preparation of the solvent: DMSO was used; medium control: culture medium was used; and negative control: in culture medium
- Stable dispersion obtained: yes
- Log Kow of the test chemical: > 6.5

DOSE RANGE FINDING ASSAY:
No dose range finder conducted. Doses were adjusted for experiment 2 based results from experiment 1. The highest test item concentration was 200 µg/mL in accordance to the OECD Guideline 442E.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: The dilutions of the test item were tested in two replicates (one labelled with anti-CD86 and one with mouse IgG1). The control groups were tested in six replicates (three labelled with anti-CD86 and three with mouse IgG1).
- Number of repetitions: 2 independent experiments
- Test chemical concentrations: experiment 1: 1, 10, 20, 50, 100 and 200 µg/mL; experiment 2: 1, 5, 10, 15, 20 and 30 µg/mL
- Application procedure: Each volume (100 µL) of the dilutions of the test item, culture medium, positive, negative and solvent control were added to the cells according to the plate template.
- Exposure time: 45 ± 3 h
- Study evaluation and decision criteria used:
For each viable condition (“%viability mean” ≥ 70%), the % of IgG1 positive cells were subtracted to the % of CD86 positive cells. The results were expressed as stimulation index (S.I.) and calculated as S.I.= ((% of CD86 treated cells - % of IgG1 treated cells)/(% of CD86 control cells - % of IgG1 control cells))x100.
The percentage of control cells (solvent/vehicle, i.e. complete medium or DMSO) was the mean of the 3 values obtained, unless one (outlier) was clearly out of the range of the other two.
Cell viability=(Number of living cells/ Total number of acquired cells)x100
If possible, the CV70 value and the EC150 value were calculated in the U-SENS™ test method by log-linear interpolation using the following equitation:
CV70 = C1+[(V1-70)/(V1-V2)*(C2-C1)]
Where:
V1: is the minimum value of cell viability over 70 %
V2: is the maximum value of cell viability below 70 %
C1 and C2 are the concentrations showing the value of cell viability V1 and V2 respectively

EC150 = C1+[(150-S.I.1)/(S.I.2-S.I.1)*(C2-C1)]
Where:
C1 is the highest concentration in µg/mL with a CD86 S.I. < 150 % (S.I.1)
C2 is the lowest concentration in µg/mL with a CD86 S.I. ≥ 150 % (S.I.2)

- Description on study acceptance criteria:
The following acceptance criteria should be met when using the U-SENS™ method:
At the end of the exposure period, the mean viability of the triplicate untreated U937 cells should be more than 90 % and no drift in CD86 expression is observed. The CD86 basal expression of untreated U937 cells had to be comprised within the range of ≥ 2 % and ≤ 25 %.
When DMSO is used as a solvent, the validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells had to be > 90 %. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250 % of the mean of the triplicate CD86 S.I. of untreated U937 cells.
The runs are considered valid if at least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6 % and < 1.5 %.
The negative control is considered valid if at least two out of the three replicates were negative (CD86 S.I. < 150 %) and non-cytotoxic (cell viability ≥ 70 %).
The positive control is considered valid if at least two out of the three replicates were positive (CD86 S.I. ≥ 150 %) and non-cytotoxic (cell viability ≥ 70 %).

SEEDING AND INCUBATION
- Seeding conditions: On the day of the experiment (U-SENS™) directly before the treatment of the cells, a volume of 100 µL with a cell density of 5  10^4 U937 cells/mL was seeded in each corresponding well of a 96-well flat bottom plate. Cells were collected in passage 8 for the first experiment and 10 for the second experiment.
- Incubation conditions: 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere
- Washing conditions: washed with approx. 100 µL of staining buffer (PBS with 5 % (w/v) FBS)

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
- Flow cytometry used: yes. flow cytometer: FACSCalibur, Becton Dickinson GmbH; using the software Cellquest Pro 6.0
- Plate used: For measurement samples were in microtubes and each was placed in a proper cytometer tube.
- cytotoxicity measurements: using Geometric mean fluorescence intensity (GeoMean(7-AAD)) for cytotoxicity (7-ADD=7-amino-actinomycin D)
- Preparation for CD54 and/or CD86 expression measurements/cell staining: All cells were transferred according to the plate template in a v-shape 96-well plate, collected by centrifugation (approx. 200  g, 5 min) and then washed with approx. 100 µL of staining buffer (PBS with 5 % (w/v) FBS). Thereafter, the cells were centrifuged, and the cell pellets were re-suspended in 100 µL staining buffer. The cells were stained with FITC-labelled anti-CD86 and mouse IgG1 (isotype control) and incubated light protected for 30 ± 5 min. on ice.

DATA EVALUATION
- Cytotoxicity assessment: see above
- Prediction model used: The Test Item was tested in at least four concentrations and in at least two independent runs to derive as single prediction (CD86 NEGATIVE or CD86 POSITIVE).
The individual conclusion of an U-SENS™ was considered NEGATIV (N) if the S.I. of CD86 is less than 150 % at all non-cytotoxic concentrations (cell viability ≥ 70 %) and if no interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) was observed.
In all other cases: S.I. of CD86 ≥ 150 % or interference is observed, the individual conclusion of an U-SENS™ run was considered POSITIVE (P).
An U-SENS™ prediction will be considered negative if the first two independent runs are negative, a third run is not necessary.
An U-SENS™ prediction will be considered positive if the first two independent runs are positive, a third run is not necessary.
Because a dose finding assay is not conducted, there is an exception if, in the first run, the S.I. of CD86 is ≥ 150% at the highest non-cytotoxic concentration only. The run will be considered as not conclusive (NC), and additional concentrations should be tested in additional runs.
In case a run will be identified as not conclusive, at least two additional runs should be conducted, and a fourth run in case runs 2 and 3 are not concordant. Follow up runs will be considered positive even if only one non-cytotoxic concentration gives a CD86 ≥ 150 %, since the concentration setting has been adjusted for the Test Item. The final prediction will be based on the majority result of the four or four individual runs.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

picrylsulfonic acid/2,4,6-trinitro-benzene-sulfonic acid (TNBS) [442E]

Positive control results:

Please refer to "Any other information on results".

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

5.9 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

1.5 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

CV70 [442E]

Value:

18.5 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

CV70 [442E]

Value:

26 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the U-SENS™ with the OECD 442E guideline recommended proficiency substances was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Results of the first run

		Microscopic evaluation
Test Group	Concen-tration [µg/mL]	Precipitation	Cytotoxicity	MEAN cell viability (%)	S.I. CD86 -IgG1 (%)	Interference IgG1 FL1 Geo Mean S.I. (%)
Medium control		no	no	97.2	93.5	92.9
	/			97.3	87.2	95.8
				97.5	119.3	111.3
	Mean			97.3	100.0	100.0
Negative control		no	no	97.6	132.3	95.9
	200			97.4	132.7	97.4
				97.7	113.5	93.8
Positive control		no	no	97.4	272.0S	108.8
	50			97.7	273.3S	106.1
				97.1	262.0S	97.4
DMSO		no	no	98.0	103.5	89.7
	/			97.3	128.1	87.7
				97.8	91.4	87.9
	Mean					88.4
Test Item	1	no	no	97.7	96.5	94.3
	10	no	no	88.2	1084.5S	108.6
	20	no	no	83.9	491.9S	124.5
	50C	no	yes	14.1	2687.6S	186.1S
	100PC	yes	yes	14.8	2602.3S	191.4S
	200PC	yes	yes	64.0	835.3S	117.9
Outcome:		positive
Calculated EC150:		1.5 µg/mL
Calculated CV70:		26.0 µg/mL

C= test groups with cytotoxic effects (cell viability < 70%) were excluded from the assessment.

S= with S.I. values of ≥ 150% .

P= Precipitations were observed

 

Table 2: Results of the second run

		Microscopic evaluation
Test Group	Concen-tration [µg/mL]	Precipitation	Cytotoxicity	MEAN cell viability (%)	S.I. CD86 -IgG1 (%)	Interference IgG1 FL1 Geo Mean S.I. (%)
Medium control		no	no	97.0	100.4	98.5
	/			97.1	124.4	102.6
				97.2	75.2	98.9
	Mean			97.1	100.0	100.0
Negative control		no	no	97.4	65.4	102.6
	200			97.0	72.9	103.1
				97.5	56.2	97.6
Positive control		no	no	96.6	263.4S	108.1
	50			96.9	294.9S	104.6
				96.6	200.5S	100.7
DMSO		no	no	97.6	73.6	93.5
	/			97.5	139.3	95.2
				97.0	72.0	84.7
	Mean			97.4	94.9	91.1
Test Item	1	no	no	97.4	113.4	101.6
	5	no	no	96.9	133.7	110.4
	10	no	no	95.9	227.8S	121.4
	15	no	no	89.4	488.6S	125.5
	20C	no	no	62.0	931.9S	143.2
	30C	no	no	5.1	2533.7S	286.9S
Outcome:		positive
Calculated EC150:		5.9 µg/mL
Calculated CV70:		18.5 µg/mL

C= test groups with cytotoxic effects (cell viability < 70%) were excluded from the assessment.

S= with S.I. values of ≥ 150% .

 

Table 3: Historical Control Data

		Min	Max	Mean	SD	n
Medium control	Mean relative viability [%]	95	98.41	124.4	0.74	48
	S.I.	75.2	124.4	100.04	10.09	48
Negative control (Lactic acid 200µg/mL)	Mean relative viability [%]	95.8	98.2	97.36	0.67	48
	S.I.	51	148	88.85	25.61	48
Positive control TNBS 50µg/mL)	Mean relative viability [%]	94.8	98.2	97.03	0.84	48
	S.I.	152	446	256.72	66.07	48
Vehicle control	Mean relative viability [%]	94.5	98.2	97.48	0.72	48
	S.I.	63	167	103.95	26.94	48

 

Interpretation of results:

other: positive for the third key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

The test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

An in vitro skin sensitization test (U-SENS™) according to OECD TG 442E was performed to assess the skin sensitization potential of the test item dissolved in DMSO when administered to U937 cells for 45 ± 3 hours. This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), and ARE-Nrf2 (luciferase test method)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The highest test item concentration was 200 μg/mL in accordance to the OECD TG 442E. For CD86 expression measurement the following concentrations of the test item were tested in the main experiments (U-SENS™): 1, 10, 20, 50, 100 and 200 μg/mL in first experiment (run) and 1, 5, 10, 15, 20 and 30 μg/mL in the second experiment. In the first experiment, cytotoxic effects (cell viability < 70 %) were observed following incubation with the test item starting with a concentration of 50 μg/mL up to the highest test concentration of 200 μg/mL, and precipitation was observed at 100 and 200 μg/mL. Therefore, the concentrations from 50 μg/mL up to 200 μg/mL were excluded from the assessment. In the second experiment cytotoxic effects were observed in the flow cytometric evaluation following incubation with the test item starting with 20 μg/mL up to the highest test concentration of 30 μg/mL. Therefore, the concentrations 20 μg/mL and 30 μg/mL were excluded from the assessment. The CV70 value of the test item was 26.0 μg/ml in the first experiment and 18.5 μg/mL in the second experiment. The test item showed interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) at 50 μg/mL and 100 μg/mL in the first experiment and at 30 μg/mL in the second experiment. This observation has no impact for the outcome of the study, since the concentrations which showed interference did not meet the cytotoxcitiy criterion and were excluded from the assessment. In the first experiment the CD86 stimulation index (S.I.) was higher or equal to 150 % after treatment with the test item between 10 μg/mL and the highest tested concentration of 200 μg/mL. Two of these concentrations (10 and 20 μg/mL) were non-cytotoxic (cell viability ≥ 70 %). In the second experiment the CD86 stimulation index (S.I.) was higher or equal to 150 % after treatment with the test item between 10 μg/mL and the highest tested concentration of 30 μg/mL. Two of these concentrations (10 and 15 μg/mL) were non-cytotoxic (cell viability ≥ 70 %). Therefore, the outcome of the U-SENS™ is considered positive for the test item. In conclusion, the test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-01-12 to 2021-01-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase LuSens test method

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: On the day of the experiment (immediately before treatment) the test item was dissolved in DMSO to prepare a stock solution with a concentration of 200 mM.
- Preparation of the test chemical serial dilutions: Dilutions were prepared by 1:2 serial dilutions from the highest soluble/dispersible concentration.
- Preparation of the positive controls: final concentration of 120 μM in treatment medium including 1 % (v/v) DMSO
- Preparation of the solvent, vehicle and negative controls: Lactic acid with a final concentration of 5000 μM in treatment medium including 1 % (v/v) DMSO; DMSO with a final concentration of 1 % (v/v) in treatment medium
- Stable dispersion obtained: yes

DOSE RANGE FINDING ASSAY:
- Highest concentration used: The highest test concentration for the dose finding assay was 2000 μM in accordance to the OECD Guideline 442D.
- Solubility in solvents: sufficiently soluble in DMSO
- Solubility in incubation medium: not specified
- Cytotoxicity assessment performed: yes
- Final concentration range selected on basis of: With reference to the CV75 parameter the highest tested concentrations in the main experiments were 24.5 μM (using a stock solution of 2.45 mM in DMSO).

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: solvent: 24, positive control: 5, negative control: 6, medium control: 12, test item: 3 per concentration
- Number of repetitions: two independent main experiments
- Test chemical concentrations: 9.9, 11.8, 14.2, 17, 20.4, 24.5 µM (main experiments)
- Application procedure: After incubation of the LuSens cells, Seeding Medium was removed and 150 μL of Treatment Medium was distributed in each well. Thereafter, 50 μL of the test item and control dilutions and the medium control (twelve replicates) were added into the corresponding wells.
- Exposure time: 48 ± 1 hour
- Study evaluation and decision criteria used:
MTT:
The quantity of formazan is presumably directly proportional to the number of viable cells, as monitored by the absorbance. The relative absorbance (= cell viability) as compared to the solvent control is calculated using this formula: relative absorbance [%]=100x ((absorbancesample – absorbanceblank)/(absorbancesolvent – absorbance blank))
absorbance sample is the MTT-absorbance reading in the sample well
absorbance blank is the MTT-absorbance reading in the blank well (no cells and treatment)
absorbance solvent control is the average MTT-absorbance reading in the wells with cells and solvent control
The arithmetic mean was calculated for each sample: test item concentrations, medium, solvent, negative and positive control.
The CV75 value, a concentration showing 75 % of LuSens cell survival (25 % cytotoxicity), is calculated by using the following equation: CV75=Conc. > 75-(((Conc. >75-Conc. <75)x(%>75-75))/(%>75-%<75))
Conc. >75 = max. measured concentration with the % of solvent control >75 % ≡ a)
Conc. <75 = min. measured concentration with the % of solvent control <75 % ≡ b)
% >75 = relative absorbance at a) in %
% <75 = relative absorbance at b) in %


Luciferase Fold Induction:
The fold induction is calculated using the following formula (OECD 442D): Fold induction= (Lsample-Lblank)/(Lsolvent-Lblank)
where
Lsample is the luminescence reading in the sample well (samples: test item concentrations, medium, negative and positive control)
Lblank is the luminescence reading in the blank well without cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent control
The arithmetic mean of the luciferase fold induction was calculated for each sample: test item concentrations, medium, negative and positive control.
- Description on study acceptance criteria:
The following acceptance criteria should be met in the LuSens test method (OECD 442D):
• The average luciferase activity induction obtained with the positive control, 120 μM EGDMA should be ≥ 2.5, and the positive control should have a relative cell viability ≥ 70 % as compared to the solvent control.
• The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells should be < 1.5 fold as compared to the average solvent control.
• The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20 % in each main experiment.
• At least three test item concentrations should have cell viability of at least 70 % relative to the solvent controls. Moreover, in case a result is to be considered negative, at least one concentration should be cytotoxic, i.e. have a cell viability < 70 %, or the maximum concentration of 2000 μM (or 2000 μg/ mL for substances with no defined MW) should have been tested.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): 9000 to 11000 LuSens cells per well (96 well microtiter plate); The passage numbers of the used LuSens cells were 5 in the cytotoxicity test and 7 and 9 in the LuSens test for the main experiments 1 and 2, respectively.
- Incubation conditions: for 24 hours ± 30 minutes at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2
- Washing conditions: At the end of the incubation period, Treatment Medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca2+/Mg2+.
- Precipitation noted: No precipitation was detected.

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: The absorption and luminescence measurement of the LuSens samples were conducted with a Multimode Reader (TriStar2 LB 942) by Berthold Technologies GmbH Co KG, Germany. The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated.
- Plate used: 96 well plate
- Lysate preparation: After washing, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 seconds per well.

DATA EVALUATION
- Cytotoxicity assessment: The CV75 value, a concentration showing 75 % of LuSens cell survival (25 % cytotoxicity), was calculated.
- Prediction model used:
If the luciferase induction is ≥ 1.5 fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥ 70 %) and at least 3 tested concentrations are non-cytotoxic, the main experiment of the LuSens prediction is considered positive.
If these conditions are met in 2 of 2 or in 2 of 3 main experiments, the LuSens prediction is considered positive, otherwise the LuSens prediction is considered negative.
A negative result obtained with a test item that does not form a stable dispersion and was not tested up to 2000 μM (or 2000 μg/mL for test items with no defined MW) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive (OECD 442D).
If no clear dose-response curve or a biphasic dose-response curve is observed, the experiment should be repeated to verify whether this is specific to the test item or due to an experimental artefact. If the biphasic response (i.e. when the threshold of 1.5 is crossed twice) is reproducible in an independent verification experiment, the lower concentration of a ≥ 1.5 induction should be reported (i.e. when the threshold of 1.5 is crossed the first time).
Mono constituent test items with a Log Pow > 7 may be insoluble in the culture medium. However, if the test item is soluble or can be stably dispersed/suspended, the LuSens test may be performed.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

EGDMA (120 M) [442D]

Positive control results:

The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 16.32; ME 2: 5.98).
The positive control had a relative cell viability ≥ 70 % as compared to the solvent control (ME 1: 128.44 %; ME 2: 99.71 %).

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: Fold induction

Value:

3.45

At concentration:

24.5 other: μM

Cell viability:

28.85

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Fold induction

Value:

3.13

At concentration:

24.5 other: μM

Cell viability:

119.51 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.33; ME 2: 0.82).
- Acceptance criteria met for positive control: yes, for details refer to positive control results.
- Acceptance criteria met for solvent control: The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 9.0 %; ME 2: 8.6 %).

Table 1: Results of the main experiment 1 (Cell viability)

Treatment group	Concentration [µM]	Absorbance (OD570)						Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
		Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank		0.013
Solvent control								0.451	0.03	0.438	100.0
Medium control								0.634	0.05	0.621	141.73
Positive Control	120	0.547	0.519	0.648	0.622	0.543		0.576	0.06	0.563	128.44
Negative Control	5000	0.513	0.599	0.556	0.562	0.557	0.411	0.533	0.07	0.520	118.68
Test Item	9.9	0.450	0.386	0.399				0.412	0.03	0.399	90.99
	11.8	0.462	0.464	0.464				0.463	0.00	0.450	102.78
	14.2	0.480	0.462	0.488				0.477	0.01	0.464	105.82
	17	0.525	0.514	0.511				0.517	0.01	0.504	114.95
	20.4	0.573	0.543	0.499				0.538	0.04	0.525	119.89
	24.5	0.598	0.516	0.496				0.537	0.05	0.524	119.51

 

Table 2: Results of the main experiment 1 (Fold induction)

Treatment group	Concentration [µM]	Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold induction
		Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank		133
Solvent control								173.1	15.6	40.1	1.00
Medium control								170.0	19.7	37.0	0.92
Positive Control	120	983	976	717	643	621		788.0	178.4	655.0	16.32
Negative Control	5000	163	185	185	170	214	200	186.2	18.8	53.2	1.33
Test Item	9.9	200	170	170				180.0	17.3	47.0	1.17
	11.8	170	192	185				182.3	11.2	49.3	1.23
	14.2	185	177	214				192.0	19.5	59.0	1.47
	17	214	185	185				194.7	16.7	61.7	1.54
	20.4	200	192	214				202.0	11.1	69.0	1.72S
	24.5	200	303	273				258.7	53.0	125.7	3.13S

S = Statistical significance (p < 0.05) was confirmed between solvent control and test item concentration via T-Test

 

Table 3 Results of the main experiment 2 (Cell viability)

Treatment group	Concentration [µM]	Absorbance (OD570)						Mean OD570	SD OD570	Mean OD570 blank corr.	Cell viability [%]
		Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank		0.013
Solvent control								0.422	0.05	0.409	100.0
Medium control								0.505	0.09	0.492	120.17
Positive Control	120	0.484	0.472	0.360	0.457	0.331		0.421	0.07	0.408	99.71
Negative Control	5000	0.434	0.548	0.514	0.494	0.495	0.405	0.482	0.05	0.469	114.59
Test Item	9.9	0.518	0.565	0.445				0.509	0.06	0.496	121.35
	11.8	0.519	0.525	0.499				0.514	0.01	0.501	122.58
	14.2	0.513	0.539	0.416				0.489	0.06	0.476	116.46
	17	0.274	0.302	0.249				0.275	0.03	0.262	64.06
	20.4	0.179	0.129	0.118				0.142	0.03	0.129	31.54
	24.5	0.140	0.136	0.117				0.131	0.01	0.118	28.85

 

Table 4: Results of the main experiment 2 (Fold induction)

Treatment group	Concentration [µM]	Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold induction
		Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank		140
Solvent control								249.4	21.3	109.4	1.00
Medium control								254.9	19.5	114.9	1.05
Positive Control	120	650	813	798	879	828		793.6	85.9	653.6	5.98
Negative Control	5000	222	207	207	244	259	236	229.2	20.9	89.2	0.82
Test Item	9.9	458	532	495				495.0	37.0	355.0	3.25S
	11.8	429	480	488				465.7	32.0	325.7	2.98 S
	14.2	421	488	517				475.3	49.2	335.3	3.07 S
	17	421	480	466				455.7	30.8	315.7	2.89
	20.4	370	451	488				436.3	60.4	296.3	2.71
	24.5	525	532	495				517.3	19.7	377.3	3.45

S = Statistical significance (p < 0.05) was confirmed between solvent control and test item concentration via T-Test

 

Table 5: Historical Control Data (2019)

	Solvent Control		Medium Control		Positive Control				Negative Control
	Luciferase induction	Relative viability [%]	Luciferase induction	Relative viability [%]	Luciferase induction		Relative viability [%]		Luciferase induction		Relative viability [%]
Min	1	100	0.69	92.17	4.28	81.51		0.60		86.87
Max	1	100	1.49	167.81	14.90	129.03		1.29		128.30
Mean	1	100	0.99	130.62	7.29	100.55		0.95		104.10
SD	0.0	0.0	0.25	17.65	3.09	15.81		0.15		9.61
n	20	20	20	20	20	20		20		20

Interpretation of results:

other: positive for the second key event of the skin sensitisation Adverse Outcome Pathway

Conclusions:

The test item activated LuSens cells under the test conditions of this study. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed according to OECD 442D. It is used to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization Adverse Outcome Pathway) of the test item. In the cytotoxicity test, cytotoxic effects (threshold of cytotoxicity: < 75 %) were observed following incubation with the test item starting with the concentration of 31.3 μM up to the highest tested concentration of 2000 μM in accordance to the OECD Guideline 442D. The CV75 value of the cytotoxicity test was calculated as 20.0 μM. The test item was tested in 2 independent main experiments. The concentrations of 9.9, 11.8, 14.2, 17.0, 20.4, 24.5 μM test item were tested in the main experiments. After treatment with the test item for 48 ± 1 hours the luciferase induction is ≥ 1.5 fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥ 70 %) and at least 3 tested concentrations are non-cytotoxic. Since these conditions are met in 2 of 2 main experiments, the LuSens prediction is considered positive. The acceptance criteria were met. The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 16.32; ME 2: 5.98). The positive control had a relative cell viability ≥ 70 % as compared to the solvent control (ME 1: 128.44 %; ME 2: 99.71 %). The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.33; ME 2: 0.82). The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 9.0 %; ME 2: 8.6 %). Furthermore, at least three test item concentrations had a cell viability of at least 70 % relative to the solvent controls. In conclusion, the test item activated LuSens cells under the test conditions of this study. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-10-08 to 2020-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

adopted 18 June 2019, corrected: 26 June 2020

Deviations:

yes

Remarks:

Please refer to "Principles of method" for details.

Principles of method if other than guideline:

Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate peptide buffer and acetonitrile (parallel dilution) instead of conducting a serial dilution as stated in the OECD 442C Guideline. This procedure was selected, since this preparation is similar to the preparation of the test item samples and controls. Furthermore, the DPRA proficiency study in the laboratory was conducted under these conditions.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

other: Synthetic Peptide Containing Cysteine (Ac-RFAACAA-OH); Synthetic Peptide Containing Lysine (Ac-RFAAKAA-OH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in approximately 20 mL aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
- Preparation of the test chemical solutions
The test item was weighed into volumetric flask and dissolved immediately before testing in acetonitrile to prepare a 100 mM stock solution.
- Preparation of the positive controls, reference controls and co-elution controls
The positive control chemical (Cinnamaldehyde) was prepared at a concentration of 100 mM in acetonitrile.
The reference control A, B and C1 samples of both peptides were prepared at a concentration of 500 μM in acetonitrile.
Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: Triplicate solutions each of the positive control and test item stock solutions were diluted with the cysteine peptide stock solution so as to prepare solutions containing 500 μM cysteine and 5 mM of Cinnamaldehyde or 5 mM of the test item. For the co-elution control, buffer solution was used in place of the cysteine stock solution.
Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls: Triplicate solutions each of the positive control and test item stock solutions were diluted with the lysine peptide stock solution so as to prepare solutions containing 500 μM lysine and 25 mM of Cinnamaldehyde or 25 mM of the test item. For the co-elution control, buffer solution was used in place of the lysine stock solution.

INCUBATION
- Incubation conditions: in the dark at 22.5 - 30 ºC for 24 ± 2 hours
- Precipitation noted: A precipitate was observed immediately upon addition of the test item solution to the peptide solution. This precipitation was observed after the incubation period of 24 ± 2 hours, too.

PREPARATION OF THE HPLC
- Analysis was performed according to the proficiency study conducted at the laboratory.
- Standard calibration curve for both Cys and Lys: The standard calibration curve linearity should have a coefficient of determination (r^2) > 0.99.
- Verification of the suitability of the HPLC for test chemical and control substances: Yes, solubility in acetonitrile was achieved.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Cysteine mean depletion: 74.8 % +/- 0.267
Lysine mean depletion: 62.1 % +/- 0.302

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

3.03 %

At concentration:

5 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean lysine depletion

Value:

0 %

At concentration:

25 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
A DPRA proficiency study in the laboratory was conducted.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not conducted
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes.
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: Please refer to "Any other information on results".

Table 1: Analytical acceptance criteria for each peptide run

	Peptide	Standard Linearity	Positive control depletion (percent points)	Reference controls (mean peptide concentration / coefficient of variation)	SD Test item depletion (percent points)
Acceptance criteria	Cysteine	r2>0.99	60.8-100 (SD <14.9%)	450 - 550 μM (CV <15%)	SD <14.9%
	Lysine	r2>0.99	40.2-69.0 (SD <11.6%)	450 - 550 μM (CV <15%)	SD <11.6%
Achieved results	Cysteine	r2=0.9993	74.8 (SD, 0.267%, n=3)	A: 523 μM (CV 0.632%, n=1) B: 509 μM (CV 0.829%, n=6) C1: 518 μM (CV 0.904%, n=3)	SD 2.37% (n=3)
	Lysine	r2=0.9997	62.1 (SD, 0.302%, n=3)	A: 509 μM (CV 0.211%, n=1) B: 491 μM (CV 2.80%, n=6) C1: 499 μM (CV 2.92%, n=3)	SD 0.00% (n=3)

 

Table 2: Depletion of peptide in the presence test material

 

	Mean peak area of reference controls	Mean peak area of peptide with test item	Mean peptide depletion by test item (%)	Mean of cysteine and lysine% depletion by test item (%)
Cysteine	Control B: 2842901 (n=6) Control C1: 2890508 (n=3)	2802890 (n=3)	3.03	1.52
Lysine	Control B: 2902948 (n=6) Control C1: 2947329 (n=3)	3025116 (n=3)	0.00

 

Historical control data (03/2018 – 08/2020)

Cysteine samples prepared at a concentration of 500 μM (376 μg/mL).

Lysine samples prepared at a concentration of 500 μM (388 μg/mL).

Table 3: Historic Data for Reference Control A

	Cysteine peptide concentration (μM)	Lysine peptide concentration (μM)
Maximum	552	548
Minimum	470	486
Mean	506	507
Number	51	51
CV (%)	2.85	2.68

 

Table 4: Historic Data for Reference Control B

	Cysteine3peptide concentration (μM)	Lysine peptide concentration (μM)
Maximum	528	533
Minimum	433	467
Mean	492	497
Number	102	102
CV (%)	3.07	2.38

 

Table 5: Historic Data for Positive Controls

	Cysteine peptide concentration (μM)	Lysine peptide concentration (μM)
Maximum	163	310
Minimum	120	178
Mean	143 a	277 b
Number	51	51
CV (%)	7.02	9.08

a: Overall mean depletion 70.9 % (n=51)

b: Overall mean depletion 44.2 % (n=51)

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item is predicted by DPRA as negative and not to be a potential skin sensitizer. However, due to precipitation the negative result cannot be used in an assessment of skin sensitisation potential.

Executive summary:

The purpose of the study (based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item. The test item was dissolved in acetonitrile when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C. Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays. With no to minimal mean depletion of both peptides (1.52 %) in the presence of the test item, the test item is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay. A precipitate was observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item. No statement can be made about how much test item remained in the solution to react with the peptide. Since this precipitation was observed before and after the incubation period of 24 ± 2 hours, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence. Therefore, the negative result cannot be used in an assessment of skin sensitisation potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

For evaluation of the skin sensitising properties of the test item a weight of evidence approach using in vitro/ in chemico experimental studies was done.

 

DPRA (reference 7.4.1-1)

The purpose of the study (based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item. The test item was dissolved in acetonitrile when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C. Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. There were no co-elution peaks in either the cysteine or lysine assays. With no to minimal mean depletion of both peptides (1.52 %) in the presence of the test item, the test item is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay. A precipitate was observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item. No statement can be made about how much test item remained in the solution to react with the peptide. Since this precipitation was observed before and after the incubation period of 24 ± 2 hours, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence. Therefore, the negative result cannot be used in an assessment of skin sensitisation potential.

 

LuSens (reference 7.4.1-2)

An in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed according to OECD TG 442D. It is used to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization Adverse Outcome Pathway) of the test item. In the cytotoxicity test, cytotoxic effects (threshold of cytotoxicity: < 75 %) were observed following incubation with the test item starting with the concentration of 31.3 μM up to the highest tested concentration of 2000 μM in accordance to the OECD Guideline 442D. The CV75 value of the cytotoxicity test was calculated as 20.0 μM. The test item was tested in 2 independent main experiments. The concentrations of 9.9, 11.8, 14.2, 17.0, 20.4, 24.5 μM test item were tested in the main experiments. After treatment with the test item for 48 ± 1 hours the luciferase induction is ≥ 1.5 fold and statistically significant compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations (cell viability ≥ 70 %) and at least 3 tested concentrations are non-cytotoxic. Since these conditions are met in 2 of 2 main experiments, the LuSens prediction is considered positive. The acceptance criteria were met. The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 16.32; ME 2: 5.98). The positive control had a relative cell viability ≥ 70 % as compared to the solvent control (ME 1: 128.44 %; ME 2: 99.71 %). The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.33; ME 2: 0.82). The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20 % in each main experiment (ME 1: 9.0 %; ME 2: 8.6 %). Furthermore, at least three test item concentrations had a cell viability of at least 70 % relative to the solvent controls. In conclusion, the test item activated LuSens cells under the test conditions of this study. Therefore, the test item is considered positive for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

U-SENS (reference 7.4.1-3)

An in vitro skin sensitization test (U-SENS™) according to OECD TG 442E was performed to assess the skin sensitization potential of the test item dissolved in DMSO when administered to U937 cells for 45 ± 3 hours. This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), and ARE-Nrf2 (luciferase test method)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The highest test item concentration was 200 μg/mL in accordance to the OECD TG 442E. For CD86 expression measurement the following concentrations of the test item were tested in the main experiments (U-SENS™): 1, 10, 20, 50, 100 and 200 μg/mL in first experiment (run) and 1, 5, 10, 15, 20 and 30 μg/mL in the second experiment. In the first experiment, cytotoxic effects (cell viability < 70 %) were observed following incubation with the test item starting with a concentration of 50 μg/mL up to the highest test concentration of 200 μg/mL, and precipitation was observed at 100 and 200 μg/mL. Therefore, the concentrations from 50 μg/mL up to 200 μg/mL were excluded from the assessment. In the second experiment cytotoxic effects were observed in the flow cytometric evaluation following incubation with the test item starting with 20 μg/mL up to the highest test concentration of 30 μg/mL. Therefore, the concentrations 20 μg/mL and 30 μg/mL were excluded from the assessment. The CV70 value of the test item was 26.0 μg/ml in the first experiment and 18.5 μg/mL in the second experiment. The test item showed interference (defined as IgG1 FL1 Geo Mean S.I. ≥ 150 %) at 50 μg/mL and 100 μg/mL in the first experiment and at 30 μg/mL in the second experiment. This observation has no impact for the outcome of the study, since the concentrations which showed interference did not meet the cytotoxcitiy criterion and were excluded from the assessment. In the first experiment the CD86 stimulation index (S.I.) was higher or equal to 150 % after treatment with the test item between 10 μg/mL and the highest tested concentration of 200 μg/mL. Two of these concentrations (10 and 20 μg/mL) were non-cytotoxic (cell viability ≥ 70 %). In the second experiment the CD86 stimulation index (S.I.) was higher or equal to 150 % after treatment with the test item between 10 μg/mL and the highest tested concentration of 30 μg/mL. Two of these concentrations (10 and 15 μg/mL) were non-cytotoxic (cell viability ≥ 70 %). Therefore, the outcome of the U-SENS™ is considered positive for the test item. In conclusion, the test item activated U-937 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

Conclusion

Based on the results of an in chemico/in vitro test strategy the test item shows no covalent binding to proteins (DPRA, OECD 442C), it activates keratinocytes (LuSens, OECD TG 422D) and activates dendritic cells (U-SENS, OECD TG442E). The negative prediction for covalent binding to proteins cannot be used in an assessment of skin sensitisation potential due to the precipitation observed. As both the second and third key event of the skin sensitisation Adverse Outcome Pathway (AOP) were considered positive, the substance is predicted to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item requires classification for Category 1 (H317: May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) No 2020/1182.