2-Propanol, 1,3-bis[(3-methyl-2-buten-1-yl)oxy]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-03 to 2021-06-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis[(3-methylbut-2-en-1-yl)oxy]propan-2-ol

EC Number:

858-735-5

Cas Number:

2337348-25-9

Molecular formula:

C13H24O3

IUPAC Name:

1,3-bis[(3-methylbut-2-en-1-yl)oxy]propan-2-ol

Test material form:

liquid

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
The test item was freshly prepared immediately prior to use. The test item was soluble in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL.

Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2 in the dose-finding assay and factor 1:1.2 in the main experiments.

The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium.

Only in the dose finding assay 2 phase separation was observed in the two highest concentrations when diluted 1:250 in cell culture medium. Sonication was used to aid solubilisation.

The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.


DOSE Groups:
1. Medium Control: cell culture medium
2. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
3. Positive Control: 4 µg/mL DNCB
4. Test Item: 8 concentrations of the test item
(dose finding assay/ main experiment)
dose finding assay 1 and 2: 1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81 µg/mL - A CV75 of 358.04 ± 55.37 µg/mL was derived in the dose finding assay
Based on the CV75, the main experiment was performed covering the following concentration steps:
main experiment 1 and 2: 429.65, 358.04, 298.37, 248.64, 207.20, 172.67, 143.89, 119.91 µg/m


APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.

Medium Control
A medium control was included in the test.

Solvent Controls
Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent previously determined.
Since the test item was solubilized in DMSO, a DMSO control served as solvent control for the test item.
Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the positive control.
The solvent controls were diluted, resulting in a final concentration of 0.2% (v/v) for DMSO.

Positive Control
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted, resulting in a final DMSO concentration of 0.2% (v/v).


MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT
Dose Finding assay:
The PI (propidium iodide) uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of lambda = 488 nm and an emission wavelength of lambda > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration.
Main experiment:
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of lambda = 488 nm and an emission wavelength of lambda = 530 nm ± 15 nm for FITC and lambda > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated.


Acceptance criteria
The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is >=150% for CD86 and >=200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not >=150% for CD86 and not >=200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.



DATA EVALUATION
- Cytotoxicity assessment
Cell viability were analysed by flow cytometry
- Prediction model used
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used. Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability = 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability = 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability = 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.
A negative result for test items with a Log KOW > 3.5 should be considered as inconclusive.

Vehicle / solvent control:

DMSO

Negative control:

other: Medium control

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

Refer to the result tables in 'Any other information on results incl. tables'

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

other: run/experiment 1 and 2

Parameter:

other: RFI CD54

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments

Remarks:

Refer to the result tables in 'Any other information on results incl. tables'

Results 2

Group:

test chemical

Run / experiment:

other: run/experiment 1 and 2

Parameter:

other: RFI CD86

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments.

Remarks:

Refer to the result tables in 'Any other information on results incl. tables'

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see below table

Any other information on results incl. tables

Discussion

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non[1]sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

 A CV75 of 358.04 ± 55.37 µg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps: 429.65, 358.04, 298.37, 248.64, 207.20, 172.66, 143.89, 119.91 µg/mL

Phase separation of the test item was observed for the two highest concentration steps in the dose-finding study 2 when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 35.4% (CD86), 38.0% (CD54) and 43.8% (isotype IgG1 control) in the first experiment and to 34.9% (CD86), 36.2% (CD54) and 47.3% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item was negative in this assay.

The controls confirmed the validity of the study for all experiments as shown in Table 6.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP

 

Results

Reactivity Check of the Cell Stock

Doubling time of the cells was monitored and found to be 32.2 h which is within the doubling time range specified by the manufacturer (30 - 55 h).

Table 2: Results of the Cell Batch Activation Test

Sample	Concentration [µg/mL]	CD86			CD54			Activated	Pass /Fail
		Cell Viability [%]	RFI	Threshold OECD TG 442E	Cell Viability [%]	RFI	Threshold OECD TG 442E	yes/no
DNCB	4 µg/mL	87	276	>150	86	413	>200	yes	pass
NiSO4	100 µg/mL	88	351	>150	87	596	>200	yes	pass
LA	1000 µg/mL	97	103	=150	98	109	=200	no	pass

 

The positive controls DNCB and NiSO₄ led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

The cell batch was accepted for further testing.

 

Solvent Finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 500 mg/mL.

 

 

Dose Finding Assay

The dose finding assay was performed using stock solutions with a concentration of 500 mg/mL (applied concentration 1000 µg/mL).

Table 3: Results of the Dose Finding Assay

Sample		Experiment 1		Experiment 2
		Concentration applied [µg/mL]	Cell Viability [%]	Concentration applied [µg/mL]	Cell Viability [%]
Medium Control	--	--	94.54	--	95.59
Solvent Control	DMSO	--	92.47	--	94.92
1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane	C8	7.81	92.73	7.81	93.61
	C7	15.63	93.79	15.63	93.08
	C6	31.25	93.24	31.25	94.57
	C5	62.50	92.72	62.50	95.86
	C4	125.00	92.35	125.00	95.63
	C3	250.00	91.21	250.00	93.70
	C2	500.00	66.94	500.00	40.44
	C1	1000.00	3.14	1000.00	1.95
Calculated CV75 [µg/mL]		397.19		318.88
Mean CV75 [µg/mL]		358.04
SD CV 75 [µg/mL]		55.37

 

 

Results CD54 and CD86 Expression

For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 19 (first experiment) and 25 (second experiment). For each experiment separately weighted samples and preparations were used.

Table 4: CD54 and CD86 Expression Experiment 1

Sample	Conc. [µg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	96.0	93.9	92.0	1144	693	490	654	203	81	53	233	141
Solvent Control	0.20%	96.7	91.9	94.6	1211	780	400	811	380	100	100	303	195
DNCB	4.00	85.4	73.8	87.9	2017	1180	395	1622	785	200	207	511	299
1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane	429.65	35.4	38.0	43.8	852	783	494	358	289	44	76	172	159
	358.04	74.7	64.8	80.5	952	806	411	541	395	67	104	232	196
	298.37	85.9	86.9	90.5	1066	724	399	667	325	82	86	267	181
	248.64	89.6	90.9	89.4	979	645	395	584	250	72	66	248	163
	207.20	92.2	90.5	94.3	1005	703	397	608	306	75	81	253	177
	172.67	92.9	92.9	93.9	905	658	393	512	265	63	70	230	167
	143.89	93.7	91.6	94.1	967	639	396	571	243	70	64	244	161
	119.91	94.8	93.9	90.3	972	605	393	579	212	71	56	247	154

 

 

 

Table 5: CD54 and CD86 Expression Experiment 2

Sample	Conc. [µg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Fluorescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	91.2	93.0	93.0	1234	742	473	761	269	79	82	261	157
Solvent Control	0.20%	92.5	90.4	92.8	1387	753	425	962	328	100	100	326	177
DNCB	4.0	80.3	77.1	76.9	3093	2070	725	2368	1345	246	410	427	286
1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane	429.65	34.9	36.2	47.3	1503	1023	517	986	506	102	154	291	198
	358.04	56.0	61.5	66.4	1587	994	426	1161	568	121	173	373	233
	298.37	84.5	82.9	86.9	1540	1033	423	1117	610	116	186	364	244
	248.64	88.2	89.3	91.3	1604	916	425	1179	491	123	150	377	216
	207.20	91.6	90.9	93.1	1591	810	401	1190	409	124	125	397	202
	172.67	92.2	92.2	94.0	1554	848	493	1061	355	110	108	315	172
	143.89	88.9	92.7	92.7	1428	835	554	874	281	91	86	258	151
	119.91	93.1	93.6	91.0	1427	979	401	1026	578	107	176	356	244

 

 

 

Acceptance Criteria

Table 6: Acceptance Criteria

Acceptance Criterion	Range	Experiment 1			pass/fail	Experiment 2			pass/fail
cell viability solvent controls [%]	>90	91.9	-	96.7	pass	90.4	-	93.0	pass
number of test dosed with viability >50% CD86	>=4	7			pass	7			pass
number of test dosed with viability >50% CD54	>=4	7			pass	7			pass
number of test dosed with viability >50% IgG1	>=4	7			pass	7			pass
RFI of positive control of CD86	>=150	200			pass	246			pass
RFI of positive control of CD54	=200	207			pass	410			pass
RFI of solvent control of CD86	<150	124			pass	126			pass
RFI of solvent control of CD54	<200	187			pass	122			pass
MFI ratio CD86/IgG1 for medium control [%]	>105	233			pass	261			pass
MFI ratio CD86/IgG1 for DMSO control [%]	>105	303			pass	326			pass
MFI ratio CD54/IgG1for medium control [%]	>105	141			pass	157			pass
MFI ratio CD54/IgG1for DMSO control [%]	>105	195			pass	177			pass

 

 Historical Data

Table 7: Historical Data

Criterion	mean	SD	N
cell viability solvent controls [%]	96.3	1.5	1554
number of test doses with viability >50%	-	-	4026
RFI of positive control of CD86	355.0	122.9	259
RFI of positive control of CD54	435.4	255.5	259
RFI of solvent control of CD86	111.1	31.3	259
RFI of solvent control of CD54	114.4	43.2	259
MFI ratio IgG1/CD86 for medium control [%]	309.0	154.2	259
MFI ratio IgG1/CD86 for DMSO control [%]	353.1	364.2	259
MFI ratio IgG1/CD54 for medium control [%]	179.0	150.3	259
MFI ratio IgG1/CD54 for DMSO control [%]	176.5	54.3	259

 

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test substance was negative in this study.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

A CV75 of 358.04 ± 55.37 µg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps: 429.65, 358.04, 298.37, 248.64, 207.20, 172.66, 143.89, 119.91 µg/mL

Phase separation of the test item was observed for the two highest concentration steps in the dose-finding study 2 when mixing the test item stock solutions with cell culture medium. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis.

Cell viability was assessed in parallel using propidium iodide staining. Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 35.4% (CD86), 38.0% (CD54) and 43.8% (isotype IgG1 control) in the first experiment and to 34.9% (CD86), 36.2% (CD54) and 47.3% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as “negative” in this assay.