2-piperidinoethanol

Endpoint summary

Description of key information

The skin sensitizing potential of the test substance was assessed using an in vitro OECD guideline testing strategy comprising the following assays:

- Direct Peptide Reactivity Assay (DPRA),

- Keratinocyte Activation Assay (LuSens), and

- Dendritic Cell Line Activation Assay (h-CLAT).

Each test was conducted under GLP according to the respective OECD guideline.

The results were as follows:

- DPRA: negative

- LuSens: positive

- h-CLAT: positive

In order to clarify the not very clear positive result of the in vitro testing strategy and to be able to sub-categorize according to the skin sensitizing potency, a Local lymph node assay in mice was conducted (OECD giudeline 429, GLP). This study gave a positive result as well.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Content: 99.9291 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: Test test substance was soluble in the vehicle (after short stirring). Because the test-substance preparation was incubated with the peptide, no analysis of the test substance in the vehicle was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM preparation in acetonitrile (considering a molecular weight of 129.20 g/mol and a purity/contents of 99.9291%). After short stirring the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in acetonitrile

Details on the study design:

TEST SYSTEM
- Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol); Lysine-(K-)containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- Source: The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K­ containing peptide). The peptide stock solution was used for preparing the calibration samples, the test-substance and control samples.

CONTROLS
- Vehicle control: acetonitrile: Set A) performance control (analyzed together with the calibration samples without incubation); Set B) Stability control (placed at the very start and ending of the sample list for HPLC analysis); Set C) for calculation of the peptide depletion (analyzed with the samples)
- Positive control: ethylene glycol dimethacrylate (EGDMA; CAS no. 97-90-5) (prepared as a 50 mM solution in acetonitrile)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide

VEHICLE
- Vehicle: acetonitrile
- Reason for choice of the vehicle: The test substance was soluble in acetonitrile (tested prior to the assay)

SAMPLE PREPARATION
- Peptide stock solutions were mixed with the test substance or positive control or vehicle control at a ration of 1:10 (C-peptide) or 1:50 (K-peptide)

EXPERIMENTAL PROCEDURE
- No. of replicates: 3 (for each peptide)
- The test substance was prepared as a 100 mM preparation in acetonitrile (considering a molecular weight of 129.20 g/mol and a purity/contents of 99.9291%). The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containg peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).
- Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis.
- Samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours
- The remaining non-depleted peptide concentration was determined by HPLC with gradient elution and UV-detection at 220 nm about 24 hours after sample preparation (for details on HPLC conditions see tab. 3). The analysis time itself did not exceed 30 hours.
- Calibration samples of known peptide concentration (dissolved in 20% acetonitrile in the respective buffer), prepared from the respective peptide stock solution used for test-substance incubation were measured before analysis of the test-substance samples with the same analytical method (for details see tab. 1)

DATA EVALUATION (for detailed formulas see "Any other information on material and methods")
Calculation of the peptide concentrations:
- For each peptide a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration. The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).
Calculation of the peptide depletion:
- The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance. When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.

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

Parameter:

other: mean peptide depletion [%]

Value:

2.33

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: see tab. 7a+b

Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

Table 6: Mean peptide depletions

	Cysteine-Peptide   mean depletion [%]          SD[%]		Lysine-Peptide   mean depletion [%]          SD[%]		mean of both depletions[%]
Test substance	4.24	0.87	0.42	0.68	2.33
PC: EGDMA in ACN	49.31	1.80	11.56	0.29	30.43

Table 7a: Historic control data of vehicle control (acetonitrile) (not including present study)

	C-peptide concentration	K-peptide concentration
	[mM]	[mM]
Min	0.436	0.475
Max	0.511	0.536
Mean	0.482	0.507
SD	0.016	0.013
n	34	32

Table 7b: Historic control data of positive control (EGDMA, 50 mM in acetonitrile) (not including present study)

	C-peptide concentration [mM]	C-peptide depletion [%]	K-peptide concentration [mM]	K-peptide depletion [%]
Min	0.080	43.32	0.395	6.79
Max	0.277	83.79	0.491	21.52
Mean	0.203	57.28	0.441	12.95
SD	0.042	7.93	0.018	3.00
n	34		32

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides.

Further, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

The mean C-peptide depletion, caused by the test substance was determined to be 4.24%.

The mean K-peptide depletion, caused by the test substance was determined to be 0.42%.

Thus, the mean peptide depletion was calculated to be 2.33%.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Remarks:

human Cell Line Activation Test (h-CLAT)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug - Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT))

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Content: 99.9291 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in culture medium. The h-CLAT was performed in an aqueous test system. Due to the use of culture medium as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared within 4 hours of application. The test substance was weighed and topped up with the chosen vehicle (culture medium) to achieve the required 2x concentration of the highest test concentration (stock solution). Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned concentrations in the culture medium. The test-substance preparations were prepared by stirring.
- Visual inspection of each dilution step was performed. Each test-substance concentration was visually inspected directly after application and after the exposure period of 24 ± 0.5 hours in order to detect test-substance precipitates.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in culture medium

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells (human monocytic leukemia cell line); obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB- 202)

CONTROLS
- Vehicle control: culture medium
- Positive control: 1-chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 µg/mL in 0.2% DMSO in culture medium
- Negative control: Lactic acid (LA, CAS no.: 50-21-5), 1000 µg/mL in culture medium
- Isotype control: In order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration and control was additionally incubated with mouse IgG1.

VEHICLE
- Vehicle: culture medium
- Reason for the vehicle: The test substance was soluble in culture medium.

SELECTION OF CONCENTRATIONS
- Cells were exposed to several concentrations of the test-substance preparation (4 µg/mL up to 5004 µg/mL corresponding to final test substance ingredient concentrations of 4 µg/mL up to 5000 µg/mL taking the purity of 99.9291% into account) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 7899 µg/mL. The highest tested concentration in the 1st main experiment was 1.2- fold of the CV75 value (1032 µg/mL). The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.

TEST CONCENTRATIONS (µg/mL; not corrected for purity/contents)
1238, 1032, 860, 716, 597, 498, 415, 346


EXPERIMENTAL PROCEDURE
- No. of replicates: 2 independent experiments with duplicates of each test-substance
- Seeding of cells: 24-well plates (500 µL of 2.0 x 10^6 cells/mL cell suspensions). Prior to use of the cells for a study, a reactivity check was performed with each new-thawed cells, as proposed in the OECD test guideline, using Nickel(II)sulfate hexahydrate, lactic acid and 1- chloro-2,4-dinitrobenzene in order to demonstrate qualification of the cells for the assay.
- Application of test substance: Treatment was performed by adding 500 µL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL.
- Exposure duration: 24 ± 0.5 hours hours under standard culture conditions (plates were sealed with semi-permeable plate sealers to prevent evaporation of the test substance)
- Each test-substance concentration was visually inspected directly after application and after the exposure period of 24 hours in order to detect test-substance precipitates.
- Cell staining and flow cytometric analysis: After visual inspection the cells were transferred into safe-lock tubes, collected by centrifugation and washed twice with 1 mL buffer (PBS (without Ca2+/Mg2+) + 0.1% BSA). Cells were incubated with 600 µL of 0.01% Globulins Chon fraction II,III at 4°C for 15 min to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquots (approximately 0.3 x 10^6 cells/180 µL/group) in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 µL working antibody solution (FITC-labelled anti-human CD86, anti- human CD54 or mouse IgG1 (isotype) antibodies; for details see tab. 1) was added to each pellet. Cell staining was performed at 4°C for 30 min in the dark. After staining the cells were washed twice with 200 µL buffer and finally re-suspended in 200 µL buffer. Before analysis in flow cytometer the cells were stained with 5 µL of PI (50 µg/mL diluted in buffer to yield a final concentration of 1.22 µg/mL PI.

DATA EVALUATION (for details on formulas see "Any other information on materials and methods")
CV75 calculation:
- The CV75-value (relative survival rate) was calculated by linear regression. This value is the substance concentration at which relative cell viability is 75% compared to the vehicle control.
Cell viability:
- Cell viability was determined by PI staining. From the independent replicates of a test substance concentration a mean was calculated.
Relative fluorescence intensity:
- Analysis of the membrane markers was performed in 10,000 living cells, determined by PI staining. Concentrations inducing viability less than 50% were not considered for further assessment of dendritic cell activation. For data analysis, the CXP software (Beckman Coulter) was used. Data evaluation was performed with mean fluorescence intensity (MFI) of chemical treated cells among the viable cells, with systematic isotype control use to quantify and remove non-specific antibody binding. After subtracting the MFI of the isotype control, the RFI of each surface marker on the treated cells as compared with the vehicle control cells was calculated. The results were expressed as relative fluorescence intensity (RFI) of % CD86 pos. or % CD54 pos. expression compared to the respective vehicle control.
EC150% and EC200% calculation:
- The concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment conducted. The calculation was performed by linear regression from the two concentrations directly above and below the EC150% / EC200% concentration.

EVALUATION CRITERIA
- A test substance is predicted to activate monocytic THP-1 cells when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 5000 µg/mL for the vehicle culture medium or 1000 µg/mL for 0.2% DMSO in culture medium) or up to the cytotoxicity limit (viability less than 90% at the highest concentration tested).
- To be relevant for evaluation, the cell viability must be more than 50% in at least four tested concentrations of an experiment.

ACCEPTANCE CRITERIA
- A tested concentration was not further evaluated when relative viability was less than 50%.
- The cell viability of vehicle control cells must yield at least 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86 < 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.
- The reactivity check of new thawed cells should produce the following result:
Positive response in CD86 and CD54 for NiSO4 and DNCB;
Negative response in CD86 and CD54 for LA.
- Positive, negative and vehicle control data should lie within the range of the historic data.

Run / experiment:

other: experiment 1

Parameter:

other: EC200% for CD54 [µg/mL]

Remarks:

concentration resulting in a RFI of 200%

Value:

710

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: experiment 2

Parameter:

other: EC200% for CD54 [µg/mL]

Remarks:

concentration resulting in a RFI of 200%

Value:

482

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 1

Parameter:

other: EC150% for CD86 [µg/mL]

Remarks:

concentration resulting in a RFI of 150%

Value:

1 032

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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
- Range of historical values: see tab. 3
- The positive and negative and vehicle control data was comparable to historic data.

The test substance was soluble in culture medium (2 x stock preparations and final concentrations).

No precipitates were noticed in any concentration after 24 hours.

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable in the 2nd experiment.

Table 1: Summary of h-CLAT Main Experiments: mean values of RFI CD86, RFI CD54 and relative viability. RFI above 150% (CD86) or 200% (CD54) with rel. viability ≥50% are indicated in bold.

1^stexperiment				2^ndexperiment
Concentration(testsubstance) [µg/mL]	RFI CD86	RFI CD54	Viability rel. viability [%]	Concentration(testsubstance) [µg/mL]	RFI CD86	RFI CD54	Viability rel. viability [%]
	mean [%]	mean [%]			mean [%]	mean [%]
346	102	130	100	346	107	143	99
415	107	124	100	415	104	147	99
498	99	104	99	498	127	212	99
597	96	121	99	597	127	268	98
716	124	204	99	716	104	190	98
860	125	194	98	860	149	394	94
1032	150	259	96	1032	134	482	86
1238	166	390	90	1238	128	341	79
VC	100	100	100	VC	100	100	100
LA 1000 µg/mL	83	123	100	LA 1000 µg/mL	68	113	99
DNCB 4 µg/mL	304	1772	70	DNCB 4 µg/mL	238	564	88

Table 2: Historic control data of LuSens. Data (not including present study)

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]		rel. viability mean [%]
Min	31	43		95		99
Max	134	196		99		101
Mean	73	114		98		100
SD	11	19		1		0
n (experiments)			230

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]		rel. viability mean[%]
Min	151	211		69		70
Max	528	2342		92		98
Mean	292	656		83		85
SD	66	308		4		4
n (experiments)			23080

Vehicle Control (culture medium)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]
Min	58	61		95
Max	144	139		99
Mean	100	100		98
SD	20	12		1
n (experiments)			230

Vehicle Control (DMSO)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]		rel.viability mean[%]
Min	39	48		90		92
Max	149	189		99		101
Mean	108	108		98		100
SD	21	18		1		1
n (experiments)			230

Table 3: Reactivity check, performed with each new-thawed cells prior to use for a study, using NiSO₄,LA, DNCB and the vehicle control

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel.viabilitymean[%]
Min	84	149	100
Max	90	169	101
Mean	73	112	100
SD	8	23	0
n (experiments)		26

Nickel(II)sulfate hexahydrate (NiSo4100 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]
Min	485	7616	84
Max	485	7616	91
Mean	391	5624	83
SD	77	1380	6
n (experiments)		26

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel.viabilitymean[%]
Min	528	932	89
Max	528	1595	93
Mean	522	1595	79
SD	86	306	9
n (experiments)		26

Conclusions:

After 24 hours of exposure to the test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-human- CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid experiments were performed.

The following results were observed:

The EC150% (the concentration resulting in a RFI of 150%) for CD86 was calculated to be 1032 μg/mL (experiment 1). The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 710 μg/mL (experiment 1) and 482 μg/mL (experiment 2), respectively.

In summary, after 24 hours of exposure to the test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Reference 3

Endpoint:

skin sensitisation: in vitro

Remarks:

ARE-Nrf2 Luciferase Test Method

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug - Oct 2018

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 Test Method)

Version / remarks:

2018

Deviations:

yes

Remarks:

Cell viability assay: 2 hours incubation with MTT reagent

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Content: 99.9291 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The stability under storage conditions over the exposure period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in 4% DMSO in culture medium. Because the test-substance preparation was applied shortly after preparation, no analysis of the test substance in the vehicle was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared within 4 hours of application. The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium (DMEM + 1% FBS) to achieve the required 4x concentration of the highest test concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate). The test-substance preparations were prepared by stirring.
- Visual inspection of each dilution step was performed.
- Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in 4 % DMSO in culture medium (DMEM + 1% FBS)

Details on the study design:

TEST SYSTEM
- Cell line: LuSens (human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany)

CONTROLS
- Vehicle control: 1% DMSO in culture medium (DMEM + 1% FBS)
- Positive control: ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 µM (= 18 µg/ml) in 1% DMSO in culture medium (DMEM + 1% FBS)
- Negative control: DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 µM (= 450 µg/ml) in 1% DMSO in culture medium (DMEM + 1% FBS)
- Blank control: culture medium (DMEM + 1% FBS) without cells
- Basal control: culture medium (DMEM + 1% FBS) with cells

VEHICLE
- Vehicle: 4% DMSO in culture medium (DMEM + 1% FBS)
- Reason for the vehicle: The test substance was soluble in 4% DMSO in culture medium (DMEM + 1% FBS)

SELECTION OF CONCENTRATIONS
- Cells were exposed to 9 concentrations of the test-substance preparation (0.5 µM up to 2001 µM corresponding to final test substance concentrations of 0.5 µM up to 2000 µM taking the purity/contents of 99.9291% into account) and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 1379 µM. Generally, the highest tested concentration in the main experiment is 1.23 fold of the CV75 value. However, as this calculated concentration is higher than the maximum concentration tested in the LuSens assay (= 2000 μM final test substance concentration) the top concentration was chosen to be 2001 μM (corresponding to 2000 μM final test substance concentration). The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.

TEST CONCENTRATIONS (µM; not corrected for purity/contents)
2001, 1668, 1390, 1158, 965, 804, 670, 558 (1st experiment)

As relative viabilities below 70% were observed in 4 concentrations of the 1st experiment, lower concentrations were tested in the 2nd and 3rd experiment in addition to the concentrations tested in the 1st experiement.

2001, 1668, 1390, 1158, 965, 804, 670, 558, 465, 388, 323, 269, 224, 187, 156, 130 (2nd and 3rd experiment)

EXPERIMENTAL PROCEDURE
- No. of replicates: 3 independent experiments with 3 replicates of each test-substance concentration in each experiment
- Seeding of cells: Cells were seeded in white (for luciferase assay) and clear (for cell viability assay) 96-well microtiter plates (120 µL of 0.83 x 10^5 cells/mL cell suspensions) in culture medium (DMEM + 10% FBS) and incubated for ca. 24 hours.
- Application of test substance: After cell adaption for 24 hours, cell culture medium was exchanged by DMEM + 1% FBS. The test substance preparations (4x concentrations) were applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%). For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.
- Exposure duration: 48 ± 1 hours under standard culture conditions (plates were sealed with semi-permeable plate sealers to prevent evaporation of the test substance)
- Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.
- Luciferase assay: After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Subsequently 200 µL of One-/Steady-Glo-preparation (= 100 µL One-/Steady-Glo- Mix and 100 µL PBS (without Ca2+/Mg2+)) per well were added and cells shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.
- Determination of cell viability: Cell culture medium was aspirated from all wells. Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium (DMEM + 1% FBS)) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

DATA EVALUATION (for details on formulas see "Any other information on materials and methods")
CV75 calculation:
- The CV75-value (relative survival rate) was calculated by linear regression. This value is the substance concentration at which relative cell viability is 75% compared to the vehicle control.
Cell viability:
- From the 3 independent replicates a mean was calculated.
Luciferase fold induction:
- From the 3 independent replicates a mean was calculated.
EC1.5 calculation:
- The concentration resulting in a positive response (1.50 fold-induction of statistical significance and viability >70%) was calculated from each experiment conducted. The calculation was performed by linear regression from the two concentrations directly above and below the EC1.50 concentration.

STATISTICAL ANALYSES
- For the statistical evaluation of luciferase fold-induction the EXCEL-function T.TEST was used.

EVALUATION CRITERIA
- A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 µM if molecular weight is applicable or 2000 µg/mL if molecular weight is not applicable) or up to the cytotoxicity limit (at least one concentration displaying viability below 70%).
- To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

ACCEPTANCE CRITERIA
- A tested concentration was not further evaluated when relative viability is less than 70%.
- The cell viability of vehicle control cells must yield at least 85%.
- The mean of the positive control EGDMA should achieve ≥2.50 fold-induction and the mean of the LA <1.50 and the mean of the viability must be ≥70%.
- The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
- The mean of the basal expression of the cells must be <1.50 fold-induction as compared to the solvent control.
- Positive, negative and vehicle control data should lie within the range of the historic data.

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 [µM]

Remarks:

1.5-fold luciferase induction

Value:

530

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: EC1.5 [µM]

Remarks:

1.5-fold luciferase induction

Value:

206

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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
- Range of historical values: see tab. 2
-The positive and negative and vehicle control data was comparable to historic data.

The test substance was soluble in 4% DMSO in culture medium (4 x stock preparations) and in 1% DMSO in culture medium (final concentrations).

No precipitates were noticed in any concentration after 48 hours.

Table 1: Summary of main experiments.

Concentration(testsubstance) [µM]	1^stexperiment
	fold induction mean	rel. viability [%] mean	t-test p-value		t-test markers
558	1.61	94	0.002		**
670	1.40	84	0.000		**
804	1.47	72	0.012		*
965	1.70	73	0.028		*
1158	2.08	61	0.003		**
1390	1.82	67	0.011		*
1668	1.81	58	0.001		**
2001	1.96	63	0.003		**
VC	1.00	100	-		-
EGDMA 90.8 µM	5.65	88	0.000		**
LA 5000 µM	0.89	108	0.011		*

Concentration (test substance) [µM]	2^ndexperiment, plate 1				Concentration(testsubstance)[µM]	2^ndexperiment, plate 2
	fold induction mean	rel. viability [%] mean	t-test p-value	t-test markers		fold induction mean	rel. viability [%] mean	t-test p-value	t-test markers
558 670 804 965 1158 1390 1668 2001	1.60 1.49 1.51 1.31 1.78 1.89 1.78 1.98	91 84 88 88 82 80 71 58	0.000 0.016 0.010 0.015 0.001 0.013 0.007 0.004	** * * * ** * ** **	130 156 187 224 269 323 388 465	1.38 1.48 1.39 1.32 1.38 1.34 1.25 1.28	88 90 100 101 98 94 97 90	0.000 0.000 0.000 0.007 0.000 0.003 0.000 0.000	** ** ** ** ** ** ** **
VC EGDMA 90.8 µM LA 5000 µM	1.00 5.60 0.80	100 84 106	- 0.000 0.000	- ** **	VC EGDMA 90.8 µM LA 5000 µM	1.00 5.44 0.80	100 80 98	- 0.000 0.001	- ** **
Concentration(testsubstance)[µM]	3^rdexperiment, plate 1				Concentration(testsubstance)[µM]	3^rdexperiment, plate 2
	fold induction mean	rel. viability [%] mean	t-test p-value	t-test markers		fold induction mean	rel. viability [%] mean	t-test p-value	t-test markers
558 670 804 965 1158 1390 1668 2001	1.61 1.52 1.73 1.62 1.49 1.79 1.52 1.71	85 72 81 72 68 61 57 60	0.001 0.016 0.010 0.014 0.027 0.004 0.013 0.007	** * ** * * ** * **	130 156 187 224 269 323 388 465	1.34 1.28 1.43 1.57 1.45 1.40 1.42 1.40	100 91 98 97 97 93 85 83	0.000 0.000 0.028 0.000 0.028 0.011 0.080 0.062	** ** * ** * *n.s. n.s.
VC EGDMA 90.8 µM LA 5000 µM	1.00 4.82 0.95	100 102 102	- 0.000 0.116	- ** n.s.	VC EGDMA 90.8 µM LA 5000 µM	1.00 5.51 0.91	100 103 110	- 0.000 0.013	- ** *

Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

Table 2: Historic control data of LuSens. (not including present study)

Negative Control (5000 µM (= 450 µg/mL))	fold induction	rel. viability [%]
Min	0.71	76
Max	1.25	140
Mean	0.94	104
SD	0.10	9
n	358

Positive Control (90.8 µM (= 18 µg/mL))	fold induction	rel. viability [%]
Min	2.83	70
Max	10.38	135
Mean	5.77	89
SD	1.45	13
n	358

Vehicle Control (1% DMSO)	fold induction	rel. viability [%]
Min	1.00	100
Max	1.00	100
Mean	1.00	100
SD	0.00	0
n	358

Basal expression	fold induction	rel. viability [%]
Min	0.61	98
Max	1.57	186
Mean	0.96	150
SD	0.15	16
n	358

Conclusions:

After 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance has a keratinocyte activating potential.

Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and

antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment, pre-tests (non-GLP) were performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 1379 μM. In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 experiments were performed.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two

independent experiments. From this it has to be concluded that the test substance has a keratinocyte activating potential.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov 2018 - 28 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland Pfalz

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Expiration date of the lot/batch: 07 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: Good solibility. Because the test-substance preparations were applied shortly after preparation, no analysis of the test substance in the vehicle was required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was produced on a weight per weight (w/w) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer, the test substance was dissolved in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH, C/O Postfach 553, NL-5800 AN Venray
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17.5 g – 20.0 g (pretest); 17.9 g – 23.7 g (main test)
- Housing: single; As group housing may increase oral exposure due to grooming of the animals and may interfere with clear observations of each individual animal, animals were single housed for the duration of the test.
- Diet: ad libitum; Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland
- Water: ad libitum; drinking water
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 13 Nov 2018 To: 26 Nov 2018

Vehicle:

propylene glycol

Remarks:

Propylene glycol was used as vehicle because good solubility of the preparation was achieved.

Concentration:

1%, 5%, 10%

The highest test substance concentration that does not induce local signs of skin irritation and/or systemic toxicity was determined in a pretest (experimental conduct in accordance with GLP, but without a GLP status).

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The test-substance preparations at different concentrations were solutions in propylene glycol (PG).
- Irritation/ear thickness measurements/erythema scores: In order to determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. Two mice were treated with test-substance concentrations of 1%, 10% and 25% each on three consecutive days. At the tested concentrations, the animals did not show relevant increases in ear weights (compared to historical vehicle values) and ear thickness measurements as indication of ear skin irritation. However, one animal of the 25% concentration showed moderate crust formation, slight scaling and lesions on the ear skin on study day 5. At the tested concentrations of 10% and 25%, the animals showed moderately increased lymph node weights. Therefore, the dose levels 1%, 5% and 10% (w/w) were selected for the main study.
- Systemic toxicity: No signs of systemic toxicity were observed in the pretest.

MAIN STUDY
- Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
- Form of application: epicutaneous
- Application volume: 25 µL per ear
- Side of application: dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Body weight determination: individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 µCi ³H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice.
- Sacrifice: on study day 5 about 5 hours after ³H-thymidine injection by cervical dislocation under Isoflurane anesthesia
- Terminal procedures:
* Determination of ear weights: Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
* Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
* Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 mL phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined by using a Casy® Counter.
* Measurement of ³H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of ³H-thymidine into the cells was measured in a β-scintillation counter.

EVALUATION OF RESULTS
- A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of ³H-thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index (SI ≥ 3.0). The biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. lymph node cell counts, lymph node weights, ear weights). The thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

For the statistical analysis of the the parameters ³H-thymidine incorporation, cell count, lymph node weight and ear weight the Wilcoxon-Test was used.

Parameter:

SI

Remarks:

for ³H-thymidine incorporation

Value:

1.15

Test group / Remarks:

1% test substance concentration

Parameter:

SI

Remarks:

for ³H-thymidine incorporation

Value:

1.75

Test group / Remarks:

5% test substance concentration

Parameter:

SI

Remarks:

for ³H-thymidine incorporation

Value:

3.21

Test group / Remarks:

10% test substance concentration

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
When applied as 10% solution in propylene glycol, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of ³H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 5% concentration was statistically significant. Concomitantly, the 10% test-substance solution induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell count. In addition, a statistically significant increase in lymph node weight was noted at the 10% concentration.
The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, statistically significant but
concentration-independent increases in ear weights were observed at the 1% and 5% concentrations.

DETAILS ON STIMULATION INDEX CALCULATION
Mean values and standard deviations of the measured parameters were calculated per test group from the individual values. The stimulation indices of ³H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.

EC3 CALCULATION
The EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI or by using the two nearest points below or above the SI.
The threshold concentration for sensitization induction was >5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for ³H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.3% and 7.7%, respectively.

CLINICAL OBSERVATIONS
No signs of systemic toxicity were noticed in all animals during general observation.
No local findings were observed during the observation period.

BODY WEIGHTS
No influence on the body weights was observed during the study.

Table 1: ³H-thymidine incorporation, cell count and lymph node weight: test group mean value, standard deviation and stimulation index

Test Group	Treatment	³H-thymidine incorporation [DPM/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	716.5	212.7	1.00
2	1% in propylene glycol	825.7	120.9	1.15
3	5% in propylene glycol	1,256.7	733.4	1.75 #
4	10% in propylene glycol	2,302.4	968.4	3.21 ##

 

Test Group	Treatment	Cell Counts [Counts/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	9,634,880	2,318,222	1.00
2	1% in propylene glycol	11,129,600	785,069	1.16
3	5% in propylene glycol	12,468,800	3,283,562	1.29
4	10% in propylene glycol	16,208,000	5,986,179	1.68 #

 

Test Group	Treatment	Lymph Node Weight [mg/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	5.4	0.9	1.00
2	1% in propylene glycol	6.1	0.8	1.13
3	5% in propylene glycol	5.9	1.0	1.10
4	10% in propylene glycol	8.0	1.8	1.49 #

 

Table 2: Ear weight: test group mean value, standard deviation and stimulation index

 

Test Group	Treatment	Ear Weight [mg/animal]
		Mean	S.D.	Stimulation Index^1
1	vehicle propylene glycol	31.6	2.0	1.00
2	1% in propylene glycol	33.6	1.4	1.06 #
3	5% in propylene glycol	35.0	2.8	1.11 #
4	10% in propylene glycol	33.9	0.9	1.07

1 test group x / test group 1 (vehicle control)

# = statistically significant for the value p ≤ 0.05

## = statistically significant for the value p ≤ 0.01

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

It is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test substance was assessed using the radioactive Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 1%, 5% and 10% (w/w) preparations of the test substance in propylene glycol (PG) or with the vehicle alone. The highest concentration was selected based on the presence of lesions on the ear skin in a pretest using a 25% test substance solution. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation

or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, 20 μCi ³H-thymidine in 250 μL sterile saline were injected

into the tail vein of the mice. About 5 hours after the ³H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated

measuring ³H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter

sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation. When applied as 10% solution in propylene glycol, the test substance induced a biologically

relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of ³H-thymidine incorporation into the cells

from the auricular lymph nodes. The increase of the 5% concentration was statistically significant. Concomitantly, the 10% test-substance solution induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell count.

In addition, a statistically significant increase in lymph node weight was noted at the 10% concentration. The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, statistically significant but concentration-independent increases in ear weights were observed at the 1% and 5% concentrations.

Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was >5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for ³H-thymidine incorporation and the EC 1.5

(estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.3% and 7.7%, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

DPRA:

In order to assess the reactivity towards peptides, synthetic cysteine (C)- or lysine (K)-containing peptides were incubated with the testsubstance for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UVdetection at 220 nm according to the OECD guideline 442C.

Therefor the test substance was dissolved at in de-ionized acetonitrile and three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide).

The test substance caused a mean C-peptide depletion of 4.24% and a mean K-peptide depletion of 0.42%.

The mean peptide depletion was calculated to be 2.33%. Thus it was concluded, that the test substance shows minimal or no chemical reactivity towards peptides.

LuSens:

In order to assess the keratinocyte activating potential, a luciferase reporter cell line (LuSens cells) was incubated with the testsubstance for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer according to the OECD guidline 442D.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated in 2 independent valid experiments to be 530 μM and 206 μM, respectively.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance has a keratinocyte activating potential.

h-CLAT:

In order to assess the potential to induce the cell membrane markers CD86 and CD54 expression the human monocytic leukemia cell line THP-1 was incubated with the testsubstance for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry according to the OECD guideline 442E.

After 24-hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid experiments were performed.

The EC150% (the concentration resulting in a RFI of 150%) for CD86 was calculated to be 1032 μg/mL (experiment 1). The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 710 μg/mL (experiment 1) and 482 μg/mL (experiment 2), respectively.

In summary, after 24 hours of exposure to the test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance 2-Piperidinoethanol induces dendritic cell activation.

Based on the results of all three tests it was concluded that the test substance is not peptide reactive and activates keratinocytes and dendritic cells. Therefore the test substance is predicted to be a skin sensitizer.

LLNA:

In order to clarify the result of the in vitro testing strategy and to sub-categorize in a strong or weak skin sensitizing potential, a Local lymph node assay in mice was conducted according to OECD guideline 429 (GLP).

Groups of 5 female CBA/CaOlaHsd mice each were treated with 1%, 5% and 10% (w/w) preparations of the test substance in propylene glycol (PG) or with the vehicle alone. The highest concentration was selected based on the presence of lesions on the ear skin in a pretest using a 25% test substance solution. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, 20 μCi ³H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the ³H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring ³H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation. When applied as 10% solution in propylene glycol, the test substance induced a biologically

relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of ³H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 5% concentration was statistically significant. Concomitantly, the 10% test-substance solution induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell count.

In addition, a statistically significant increase in lymph node weight was noted at the 10% concentration. The test-substance concentrations did not cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, statistically significant but concentration-independent increases in ear weights were observed at the 1% and 5% concentrations. Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for ³H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 9.3% and 7.7%, respectively.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be classified for skin sensitization (category 1B) under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.