Phenylacetylene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 17, 2022 to March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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:

June 25, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

Non-animal testing is the default requirement for skin sensitisation.

Test material

Test material information

Constituent 1

Reference substance name:

Phenylacetylene

EC Number:

208-645-1

EC Name:

Phenylacetylene

Cas Number:

536-74-3

Molecular formula:

C8H6

IUPAC Name:

ethynylbenzene

Test material form:

liquid

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

PRELIMINARY STUDY : Cytotoxicity assays
The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50%, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods.
Eight concentrations of test item were prepared by a two-fold serial dilution from a maximum final concentration of 1000 µg/mL or lower, depending on its solubility limit.
In the day of testing, cells harvested from culture flask were suspended with fresh culture medium at 2 x 10^6 cells/mL. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 µL (1 x 10^6 cells/well) with various concentrations of test item (1:1 ratio) for 24±0.5 hours at 37 °C under 5 % CO2. After treatment, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with phosphate-buffered containing 0.1% (w/v) bovine serum albumin identified as Fb .Cells were stained with 7-AAD (5 µg/mL final concentration). Then cells were analysed with flow cytometry using GUAVA (Merck Millipore, France) and InCyte software to measure cell viability. The living cells (7-AAD-) gate was set in the 7 AAD negative area. 10^4 7-AAD- cells were counted as the living population.

Note: Before staining with 7-AAD, the two additional steps of washing the cells with Fb (recommended in the guidelines) is not performed (validated in internal research).

MAIN STUDY : Activation test
In case of non-toxic concentration for the top dose used in preliminary test, the maximum concentration selected for activation test must not exceed 1000 µg/mL when the test item is dissolved or stably dispersed in Ethanol or DMSO, and 5000 µg/mL when the test item is dissolved in a saline vehicle.
In case of product cytotoxicity, the eight final test item concentrations are selected approximately according to the calculated CV75.
The range of concentrations was adjusted for activation test 2 and 3 (if necessary) depending on results obtained in previous experiments, to calculate the minimum induction threshold. Each activation test was performed on eight concentrations.

THP-1 cells (provided from American Type Culture Collection (batch 61077351)) were plated at 1*106 cells/mL/well in 24 well plates and treated for 24±0.5 hours at 37±1°C under 5±1% CO2 with selected test item concentrations. After treatment cells were washed twice with Fb.
Note: After washing, blocking the cells with Fb buffer containing 0.01% (w/v) globulin (recommended in the guidelines) was not performed due to low expression of receptors FcR by the cells THP-1.

Then cells were stained for 30 min at about 4°C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse IgG1. Using the manufacturer’s recommended dilutions, cells were incubated with above mAbs at 6 µL/3*105 cells /50µL for the anti-human CD86 mAb, and 3 µL/3*105 cells /50µL for the anti-human CD54 mAb. FITC labelled-mouse IgG1 was used as an isotype control at a dilution of 3 µL/3*105 cells /50µL. Then, the cells were stained also with 7-AAD for at least 30 min at about 4°C. After washing and resuspension with Fb, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA (Merck Millipore, France) and InCyte software, on 10000 living cells.
Note: Before staining with 7-AAD, the cells were washed once with Fb instead of twice as recommended in the guidelines (validated in internal research).

PREPARATION OF TEST SOLUTIONS
- Solubility of the test item in solvents:
The test item was soluble in Ethanol, the highest solubilized concentration was 1000 µg/mL. Based on the solubility test, the doses range for cytotoxicity test was the following (with two fold dilution factor): from 7.81 to 1000 µg/mL.

- Vehicle control:
As the maximal dose for this test item (500 mg/mL) was obtained in the Ethanol, then, the vehicle control for all of the assays in this study was Ethanol, provided by Sigma.

- Preparation of the positive controls:
*2,4-dinitrochlorobenzene (DNCB) was prepared in DMSO and the final treatment concentration was 4 μg/mL in the plate. The DNCB was supplied by Sigma.
*Nickel sulfate (NiSO4) was prepared in PBS and the final treatment concentration was 100 μg/mL in the plate. The NiSO4 was supplied by Sigma.

- Preparation of the negative control:
The negative control was Lactic acid (LA), prepared in 0.9% NaCl and the final treatment concentration was 1000 μg/mL in the plate. The LA was supplied by Sigma.

DATA EVALUATION
- Cytotoxicity assessment :
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity) is calculated by log-linear interpolation using the following equation:
Log CV75 = [(75-c) x Log (b) – (75-a) x Log (d)] / (a – c)
a: minimum value of cell viability over 75%
c: maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively

- Analysis of results :
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test article-treated cells were calculated according to the following equation:
RFI = (MFI of test item-treated cells – MFI of test item-treated isotype control cells / MFI of vehicle control cells – MFI of vehicle isotype control cells) x100

When the cell viability was less than 50%, RFI was not exploited because of the diffuse labelling of cytoplasmic structures that are generated following cell membrane destruction.

- Prediction model used:
If two or three independent experiments, at any dose exceed the positive criteria: The RFI of CD86 ≥ 150% or the RFI of CD54 ≥ 200% of their respective vehicle control, the test item may be identified as a sensitizer. Otherwise it is identified as a non-sensitizer.

- Calculation of Effective Concentration (EC) Value :
For test articles predicted as POSITIVE, two EC values, the EC150 for CD86 and the EC200 for CD54, , i.e. the concentration at which the test item induced a RFI of 150 or 200, may be determined, are calculated as follows:
EC150 (for CD86) = Bconcentration + [(150 – BRFI) / (ARFI – BRFI) x (Aconcentration – Bconcentration)]
EC200 (for CD54) = Bconcentration + [(200 – BRFI) / (ARFI – BRFI) x (Aconcentration– Bconcentration)]

Where: A concentration is the lowest concentration in µg/mL with RFI >150 (CD86) or 200 (CD54) ; Bconcentration is the highest concentration in µg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI >150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54).

The ECs are calculated for each experiment. If three experiments are performed, the final EC150/EC200 values are then determined as the median value of the ECs calculated from the three independent runs. When only two experiments are performed or two of three independent runs meet the criteria for positivity, the highest EC150 or EC200 of the two calculated values are adopted.

- Determination of MIT (Minimum Induction Threshold):
The MIT is determined as the smallest of either EC150 or EC200.

ASSAY ACCEPTANCE CRITERIA
-In the positive control (DNCB) (performed in all activation experiments) and (NiSO4) (performed only in an activation experiment):
* RFI values of both CD86 and CD54 must be over the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
* The cell viability must be more than 50%.
-In the negative control (LA) (performed only in an activation experiment):
* RFI values of both CD86 and CD54 must be under the positive criteria (CD54 < 200% and CD86 < 150%).
* The cell viability must be more than 50%.
-In the vehicle control (medium, 0.9% NaCl, DMSO, Ethanol etc.):
* Cell viability must be more than 90%.
* In the solvent/vehicle control, RFI values of both CD86 and CD54 must not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) compared to the medium control.
* The MFI ratio of both CD86 and CD54 to isotype control must be > 105%.
-In the test item:
* Cell viability must be more than 50% in at least four tested concentrations in each run.

Vehicle / solvent control:

other: Ethanol

Negative control:

DL-Lactic acid

Positive control:

other: 2,4-dinitrochlorobenzene (DNCB) and Nickel sulfate (NiSO4)

Results and discussion

Positive control results:

Results of positive controls met the acceptance criteria defined for this method.

In vitro / in chemico

Results

Key result

Group:

test chemical

Run / experiment:

other: Preliminary cytotoxicity test

Parameter:

CV75 [442D and 442E]

Cell viability:

>75% in all tested concentrations

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Due to % cell viability>97% in all tested concentrations : non cytotoxic

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test system was checked after thawing. DNCB and nickel sulphate produce a positive response of both CD86 and CD54 and lactic acid produce a negative response of both CD86 and CD54, in experiment 1 and experiment 2.

ACCEPTANCE OF RESULTS:
Test system was validated as all acceptability criteria were fulfilled:
-The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent experiment.
-For the solvent/vehicle controls, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
-For both medium and solvent/vehicle controls, the ratio of both markers CD86 and CD54 to isotype control was >105% on all occasions.
-For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent experiment.
-For the test article, the cell viability was more than 50% in all tested concentrations in each independent experiment.

Any other information on results incl. tables

Main test summary results

Relative Fluorescence Intensity (RFI) of CD54/86 expressions are presented in table 7.4.1.1:

Sample	Dose Level (µg/mL)	Experiment 1			Experiment 2
		CD54	CD86	Cell Viability (%)	CD54	CD86	Cell Viability (%)
Ethanol		97	88	97.3	93	102	98.1
RPMI (Rowell Park Memorial Institute Medium)		100	100	97.5	100	100	97.8
DMSO	-	143	124	96.6	134	147	97.8
DNCB	4	1417	466	80.4	1283	431	82.5
Test article (vehicle: 0.2% Ethanol in RPMI)	7.81	90	101	97.3	97	92	98.2
	15.6	90	89	97.8	85	97	98.3
	31.3	95	93	97.6	85	83	97.9
	62.5	88	101	97.7	103	82	98.1
	125	113	109	97.2	106	106	97.8
	250	109	104	97.0	106	94	97.8
	500	105	93	96.5	106	89	97.2
	1000	108	108	96.3	100	93	95.7

Quality control of the test system

Results (obtained in the experiment 1) are summarized in the following tables:

 

Table 7.4.1.2: Cell viability and ratio of both markers CD54 and CD86 to isotype control obtained for vehicles and controls

Sample	Cell viability (%)		Ratio CD54/IgG		Ratio CD86/IgG
	Criteria	Results	Criteria	Results	Criteria	Results
Ethanol (0.2%, v/v in RPMI)	>90%	97.3	>105	121	>105	193
RPMI	>90%	97.5	>105	120	>105	198
	>90%	97.3	>105	122	>105	216
DMSO (0.2%, v/v in RPMI)	>90%	96.6	>105	132	>105	234
NiSO4	>50%	84.5	/	/	/	/
DNCB	>50%	80.4	/	/	/	/
LA	>50%	97.0	/	/	/	/

Table 7.4.1.3: RFI values obtained for vehicles and controls

Sample	RFI
	Criteria	Results CD54	Results CD86
Ethanol (0.2%, v/v in RPMI)	<200 (CD54) and <150 (CD86)	97	88
0.9% NaCl (1%, v/v in RPMI)	<200 (CD54) and <150 (CD86)	104	113
DMSO (0.2%, v/v in RPMI)	<200 (CD54) and <150 (CD86)	143	124
NiSO4	≥200 (CD54) and ≥150 (CD86)	5613	316
DNCB	≥200 (CD54) and ≥150 (CD86)	1417	466
LA	<200 (CD54) and <150 (CD86)	116	56

Results (obtained in the experiment 2) are summarized in the following tables:

 

Table 7.4.1.4: Cell viability and ratio of both markers CD54 and CD86 to isotype control obtained for vehicles and controls

Sample	Cell viability (%)		Ratio CD54/IgG		Ratio CD86/IgG
	Criteria	Results	Criteria	Results	Criteria	Results
Ethanol (0.2%, v/v in RPMI)	>90%	98.1	>105	123	>105	212
RPMI	>90%	97.8	>105	124	>105	205
	>90%	97.8	>105	134	>105	265
DNCB	>50%	82.5	/	/	/	/

Table 7.4.5: RFI values obtained for vehicles and controls

Sample	RFI
	Criteria	Results CD54	Results CD86
Ethanol (0.2%, v/v in RPMI)	<200 (CD54) and <150 (CD86)	93	102
DMSO (0.2%, v/v in RPMI)	<200 (CD54) and <150 (CD86)	134	147
DNCB	≥200 (CD54) and ≥150 (CD86)	1283	431

According to these results, the test system was valid for these experiments. 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test article Phenylacetylene, was considered to be negative in the human Cell Line activation Test. Therefore, the test item has not to be classified as skin sensitiser according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

The study was conducted to investigate the potential of Phenylacetylene (batch N°LTWK2021-02) to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E and in compliance with GLP.

A solubility of the test item in a suitable solvent was assessed before performing the assay. The tes item was soluble in Ethanol, the highest solubilized concentration was 1000 µg/mL. 

 

Firstly cytotoxicity profile of the test item was assessed with the 7-AAD dye. Based on the solubility test, the doses range for cytotoxicity test was the following (with two fold dilution factor): from 7.81 to 1000 µg/mL. No cytotoxicity was induced on THP-1 cells by the test item (% cell viability>75% in all tested concentrations) and no CV75 value could be determined. Under the assay conditions, no reproducible "increase" of the CD54/86 expression compared with the vehicle control was noticed, for the test item.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

 

The test article Phenylacetylene (batch N°LTWK2021-02) was considered to be negative in the human Cell Line activation Test. Therefore, the test item has not to be classified as skin sensitiser according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.