4-(5-Propyl-tetrahydropyran-2-yl)-phenol

Endpoint summary

Description of key information

OECD 442C: no data

OECD 442D: positive

OECD 442E: positive

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:

14 Sep. 2021 - 09. Dec 2021

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

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
On the day of the experiment (immediately before treatment) the test item was dissolved in DMSO to prepare a stock solution. The highest test concentration for the dose finding assay was 2000 µM in accordance with the OECD guideline 442D.
For the MTT test (dose finding assay) twelve concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest concentration. With reference to the CV75 parameter, the highest tested concentrations in the main experiments were 50 µM.


DOSE RANGE FINDING ASSAY:
The doses investigated in the main experiment (LuSens) were determined with a MTT test. The MTT test is based on the cleavage of the yellow tetrazolium salt MTT [=3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid] to form a blue-violet formazan dye by MTT reduction. One cytotoxicity experiment (dose finding assay) was performed to obtain a CV75.


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates 3
- Number of repetitions 2
- Test chemical concentrations 20.1, 24.1, 28.9, 34.7, 41.7, 50.0 µM
- Application procedure
For the treatment of the cells in the main experiments, a stock solution of the test item and the controls were prepared. The solvent control DMSO (24 replicates), the positive control (five replicates) and the negative control (six replicates), as well as the test item concentrations (each three replicates) were subsequently diluted 1:25 in treatment medium. The treatment medium control (twelve replicates) was used undiluted. 24 h ± 30 min after seeding of the cells, seeding medium were removed and 150 µL of treatment medium were distributed in each well. Thereafter, 50 µL of the test item and control dilutions and the medium control were added into the corresponding wells. At the end of the incubation period of 48 ± 1 h under standard cell culture conditions, the cell cultures were microscopically evaluated for morphological alterations or precipitation.

- Exposure time 48 ± 1 h
- Study evaluation and decision criteria 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).
- Description on study acceptance criteria
• 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.

- Other:

SEEDING AND INCUBATION
Between 4 and 6 x 10^5 or 6 and 8 x 10^5 cells were seeded in 15 mL cultivation medium and pre-cultured at least twice a week in culture flasks (80-90% confluent). The cell density between approximately 80-90% should not be exceeded.
After microscopic assessment of the cells, the cells were washed at least twice with 10 mL Ca2+/Mg2+ free DPBS including EDTA. Thereafter, the cells were trypsinated with 1 to 2 mL 0.05% Trypsin/EDTA for approximately 5 min at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2. The cells were resuspended in 10 mL cultivation medium to neutralise the trypsin.
For seeding of the cells, the cultivation medium was removed, and the cells were transferred into seeding medium. Each treatment well of a 96 well microtiter plate was seeded with 100 µL cell suspension (9000 to 11000 LuSens cells per well), except the well of the blank control. The cells were incubated for 24 h ± 30 min under standard cell culture conditions.

LUCIFERASE ACTIVITY MEASUREMENTS
The Steady-Glo® mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca2+/Mg2+) with one part of Steady-Glo® mix.
At the end of the incubation period (48 ± 1 h), the treatment medium was removed from the wells and the cells were washed at least twice with 200 µL DPBS including Ca2+/Mg2+. Thereafter, 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 sec per well.


DATA EVALUATION
The doses investigated in the main experiment (LuSens) were determined with a MTT test. The MTT test is based on the cleavage of the yellow tetrazolium salt MTT [=3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid] to form a blue-violet formazan dye by MTT reduction. One cytotoxicity experiment (dose finding assay) was performed to obtain a CV75. The MTT working solution consists of two components, the MTT stock solution (5 mg/mL MTT in Ca2+/Mg2+ free PBS) and treatment medium. Both components were mixed immediately before application at a ratio of 1:10. For the treatment of the cells in the dose finding assay, a stock solution of the test item and the controls were prepared. The test item was dissolved in the solvent and 1:2 serially diluted in the solvent to obtain the desired test item concentrations (twelve concentrations). All values stated in the report are rounded values. The solvent (twelve replicates), the positive (two replicates) and the negative (three replicates) controls as well as the test item concentrations (each three replicates) were subsequently diluted 1:25 in treatment medium. The medium control (six replicates) was used undiluted.
24 h ± 30 min after seeding of the cells, the seeding medium was removed from the wells. Thereafter, 150 µL treatment medium were added per well and 50 µL of the test item dilutions, the solvent, negative and positive controls and the medium control were added to the wells, respectively. At the end of the incubation period of 48 ± 1 h under standard cell culture conditions, the cell cultures were microscopically evaluated for morphological alterations or precipitation.
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+. Thereafter, 200 µL of the MTT working solution were added to each treatment well and the cells were incubated for 3 hours ± 30 min under standard cell culture conditions. After rinsing the MTT working solution, the cells of each well were treated with 100 µL MTT lysis agent (isopropanol with 0.04 N HCl) for at least 30 min, while gently shaking. Thereafter, the microplate was transferred to a Multimode Reader equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).
The quantity of formazan is presumably directly proportional to the number of viable cells, as monitored by the absorbance.
- Other:

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: 7.73; ME 2: 7.44) and statistically significant.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: luciferase induction

Value:

614 %

At concentration:

0.05 mM

Cell viability:

96.28 % @ 50 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD86>200 [442E]

Value:

373 %

At concentration:

0.05 mM

Cell viability:

72.77 % @ 50 µM

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:

Acceptance criteria:

- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥2.5 (ME 1: 7.73; ME 2: 7.44) and statistically significant.
- The positive control had a relative cell viability ≥70% as compared to the solvent control (ME 1: 117.31%; ME 2: 118.83%).
- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid (ME 1: 1.34; ME 2: 1.13), as well as the basal expression of untreated cells was <1.5-fold as compared to the average solvent control (ME 1: 1.16; ME 2: 1.05).
- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 8.9%; ME 2: 6.0%).
- At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls.

The test mets the acceptance criteria.

Results of the main experiment 1 (cell viability)

Treatment group	Concentration					Absorbance (OD570)						Mean OD570	SD OD570	Mean OD570blank corr.	Cell viability [%]
						Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank						0.022
Solvent control												0.472	0.04	0.450	100.00
Medium control												0.556	0.05	0.534	118.52
Positive control		120		µM		0.522	0.508	0.616	0.478	0.628		0.550	0.07	0.528	117.31
Negative control		5000		µM		0.510	0.397	0.509	0.455	0.491	0.472	0.472	0.04	0.450	99.98
Test item	C1	20.1		µM		0.568	0.443	0.505				0.505	0.06	0.483	107.31
	C2	24.1		µM		0.581	0.565	0.563				0.570	0.01	0.548	121.59
	C3	28.9		µM		0.585	0.546	0.614				0.582	0.03	0.560	124.26
	C4	34.7		µM		0.508	0.532	0.515				0.518	0.01	0.496	110.19
	C5	41.7		µM		0.492	0.501	0.503				0.499	0.01	0.477	105.83
	C6	50.0		µM		0.426	0.431	0.510				0.456	0.05	0.434	96.28

Results of the main experiment 1 (fold induction)

Treatment group	Concentration					Luminescence						Mean luminescence	SD luminescence	Mean luminescence blank corr.	Fold induction	p-value, if significant (two sample t-test)
						Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank						118
Solvent control												216.1	19.13	98.1	1.00
Medium control												232.2	15.49	114.2	1.16
Positive control		120		µM		732	909	879	939	924		876.6	83.82	758.6	7.73	0.000
Negative control		5000		µM		229	281	236	236	251	266	249.8	20.25	131.8	1.34
Test item	C1	20.1		µM		480	510	480				490.0	17.32	372.0	3.79	0.006
	C2	24.1		µM		406	384	392				394.0	11.14	276.0	2.81	0.000
	C3	28.9		µM		576	517	554				549.0	29.82	431.0	4.39	0.000
	C4	34.7		µM		569	488	621				559.3	67.02	441.3	4.50	0.000
	C5	41.7		µM		628	665	621				638.0	23.64	520.0	5.30	0.000
	C6	50.0		µM		761	702	695				719.3	36.25	601.3	6.13	0.000

Results of the main experiment 2 (cell viability)

Treatment group	Concentration					Absorbance (OD570)						Mean OD570	SD OD570	Mean OD570blank corr.	Cell viability [%]
						Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank						0.014
Solvent control												0.491	0.05	0.477	100.00
Medium control												0.631	0.09	0.617	129.28
Positive control		120		µM		0.573	0.789	0.604	0.486	0.454		0.581	0.13	0.567	118.83
Negative control		5000		µM		0.609	0.483	0.533	0.611	0.603	0.707	0.591	0.08	0.577	120.88
Test item	C1	20.1		µM		0.476	0.330	0.453				0.420	0.08	0.406	84.99
	C2	24.1		µM		0.486	0.366	0.419				0.424	0.06	0.410	85.82
	C3	28.9		µM		0.492	0.374	0.436				0.434	0.06	0.420	87.99
	C4	34.7		µM		0.477	0.356	0.459				0.431	0.07	0.417	87.29
	C5	41.7		µM		0.460	0.392	0.413				0.422	0.03	0.408	85.41
	C6	50.0		µM		0.408	0.319	0.357				0.361	0.04	0.347	72.77

Results of the main experiment 2 (fold induction)

Treatment group	Concentration					Luminescence						Mean luminescence	SD luminescence	Mean luminescence blank corr.	Fold induction	p-value, if significant (two sample t-test)
						Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank						126
Solvent control												209.2	12.51	83.2	1.00
Medium control												213.2	18.58	87.2	1.05
Positive control		120		µM		835	739	769	717	665		745.0	63.04	619.0	7.44	0.000
Negative control		5000		µM		229	192	185	214	251	251	220.3	28.45	94.3	1.13
Test item	C1	20.1		µM		310	325	347				327.3	18.61	201.3	2.42	0.000
	C2	24.1		µM		370	333	340				347.7	19.66	221.7	2.67	0.000
	C3	28.9		µM		495	347	392				411.3	75.87	285.3	3.43	0.030
	C4	34.7		µM		384	362	392				379.3	15.53	253.3	3.05	0.000
	C5	41.7		µM		377	333	370				360.0	23.64	234.0	2.81	0.025
	C6	50.0		µM		495	406	406				435.7	51.38	309.7	3.72	0.005

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

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 AOP.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 Sep. 2021 to 16 Dec. 2021

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:

https://doi.org/10.1787/9789264264359-en.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

U937 cell line activation test (U-SENS™)

Details of test system:

U-937 cell line [442E]

Details on the study design:

The U937 Line Activation Test (U-SENS™) addresses the third key event by measuring phenotypic changes, such as CD86 expression on dendritic cells. The human histiocytic leukemia cell line U937 is used as surrogate for human myeloid dendritic cells since these cells show also enhanced CD86 expression when treated with sensitisers.
The purpose of the U-SENS™ test is to assess the skin sensitising potential of the test item in an appropriate solvent (DMSO or culture medium) when administered to U937 cells.
This U-SENS™ 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 AOP for the assessment of the skin sensitisation potential of chemicals.
The technical proficiency of the U-SENS™ with the OECD 442E guideline recommended proficiency substances was demonstrated.

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:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.55 in experiment 1; 6.13 in experiment 2)

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: CD86 stimulation index (S.I.)

Value:

150 %

At concentration:

10 other: µg/mL

Cell viability:

In the first experiment, cytotoxic effects (cell viability <70%) were observed following incubation with the test material starting with a concentration of 50 µg/mL up to the highest tested concentration. Precipitation was observed at 200 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:

other: CD86 stimulation index (S.I.)

Value:

150 %

At concentration:

10 other: µg/mL

Cell viability:

In the second experiment, cytotoxic effects were observed following incubation with the test material concentration of 50 µg/mL.

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:

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 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. is 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 fall within the range of ≥0.6% and <1.5%.
• The negative control is considered valid if at least two out of the three replicates are 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 are positive (CD86 S.I. ≥150%) and non-cytotoxic (cell viability ≥70%).

Test group	Concentration [µg/mL]	Microscopic evaluation		Mean cell viability [%]	S.I. CD86 -IgG1 [%]	Interference IgG1 FL1 Geo Mean S.I. [%]
		Precipitation	Cytotoxicity
Medium control		no	no	94.1	111.2	104.5
	/			94.5	103.5	99.9
				94.8	85.2	95.7
	Mean			94.5	100.0	100.0
Negative control		no	no	93.7	97.3	100.1
	200			93.8	104.3	100.3
				94.6	85.7	98.1
Positive control		no	no	93.7	291.7	117.0
	50			93.4	264.5	113.1
				93.9	261.5	105.6
DMSO		no	no	94.3	140.2	83.3
	/			94.6	117.5	86.2
				95.2	88.1	83.3
	Mean			94.7	115.2	84.3
Test item	1	no	no	94.4	104.5	104.9
	2	no	no	93.6	122.3	113.0
	5	no	no	92.9	177.3	137.3
	10	no	no	89.7	249.2	169.0
	20	no	no	85.3	405.5	190.9
	50	no	yes	14.4	1060.6	195.9
Outcome:		positive
Calculated EC150:		3.5 µg/mL
Calculated CV70:		26.5 µg/mL

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The provided information provides evidence for classification according to the EU Regulation (EC) No 1272/2008 or any updates on Classification,Labelling and Packaging of Substances and Mixtures.