Cinnamyl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.07.2016-09.06.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E h-CLAT

Version / remarks:

Dec 2015

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 by XTT test instead of flow cytometry. The reactivity check for the qualification of the cells conducted with positive control (DNCB) and negative control (lactic acid) but without nickel sulfate.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessen Ministerium für Umwelt, Klimatschutz, Landwirtschaft und Verbraucherschutz; Wiesbaden.

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Cinnamyl acetate

EC Number:

203-121-9

EC Name:

Cinnamyl acetate

Cas Number:

103-54-8

Molecular formula:

C11H12O2

IUPAC Name:

cinnamyl acetate

Specific details on test material used for the study:

purity: 99.3

In vitro test system

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: ATCC, #TIB-202 (provided by LGC Standards GmbH, Germany)

TEST-SUBSTANCE PREPARATION
- Concentrations: 1st, 2nd and 3rd experiment: 75.4, 90.4, 108.5, 130.2, 156.3, 187.5, 225 and 270 μg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Vehicle: culture medium
- Reason for vehicle: The h-CLAT was performed in an aqueous test system.

CONTROLS
- Vehicle control (VC): DMSO in culture medium, final concentration 0.2%
- Isotype control:10 μL FITC-labelled anti-CD86, CD54 antibody or mouse IgG1
- Positive control: DNCB final concentration: 2 and 3 μg/mL
MEDIUM
- Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamineand appropriate antibiotics (100U/mL of penicillin and 100μg/mL of streptomycin)
- FACS Buffer: PBS with 0.1% (w/v) BSA

EXPERIMENTAL PROCEDURE
- Replicates:7
- Experiments: 3
- Exposure period: 24 ± 1 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamineand appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) (<= 30 passage)

ANALYSIS
- FACS: cell staining and flow cytometric analysis

DATA EVALUATION
- Relative fluorescence intensity: RFI (%) = ( MFI of chemical-treated cells - MFI of chemical-treated
isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells) * 100
- Relative cell viability: % relative cell viability = (cell viability of test substance treated cells / mean cell
viability of vehicle control treated cells) * 100

ACCEPTANCE CRITERIA
- In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%
- In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%)
- For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%
- For the test item resulting in negative outcome, the cell viability at the1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5mg/mL in saline, 1mg/mL in DMSO or the highest soluble concentration will be used as the maximal test concentration instead of CV75-based concentration, the data for test item are accepted independent by the cell viability.)
- The cell viability of at least 4 concentrations in each experiment should be ≥50%.

EVALUATION RESULTS
- The test item is tested in 2 independent runs.
- If the RFI of CD86 is ≥ 150% and/or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be positive in the h-CLAT. Otherwise it is considered to be negative in the h-CLAT.
- In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any concentration in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test item is considered to be positive.
- Otherwise it is considered to be negative.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: CD86

Parameter:

other: RFI

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: As this slight increase was observed only in 1 out of 3 independent runs, the test item is considered negative in the h-CLAT.

Results 2

Key result

Run / experiment:

other: CD54

Parameter:

other: RFI

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: As this slight increase was observed only in 1 out of 3 independent runs, the test item is considered negative in the h-CLAT.

Any other information on results incl. tables

Results of the first h-CLAT run for the Test Item:

	Concentration (μg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control		100.0	100.0	100.0
DMSO Control		100.0	100.0	100.0
Positive Control (DNCB)	2.0	967.1*	504#	87.6
	3.0	1230*	402.9#	70.2
Test Item	75.4	120.6	96.6	88.2
	90.4	114.3	119.0	95.0
	108.5	139.7	108.0	93.0
	130.2	154.0	117.2	88.4
	156.3	187.3	135.1	84.3
	187.5	214.3*	153.4#	91.4
	225^P	271.4*	175.9#	88.5
	270^P	395.2*	223.6#	76.6

^Pprecipitation/phase separation (oily droplets), are excluded from the evaluation

*RFI value of CD54 exceeded the positive criterion (CD54 ≥ 200%)

#RFI value of CD86 exceeded the positive criterion (CD86 ≥ 150%)

Results of the second h-CLAT run repetition for the Test Item:

	Concentration (μg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control		100.0	100.0	100.0
DMSO Control		100.0	100.0	100.0
Positive Control (DNCB)	2.0	246.4*	236.5#	74.7
	3.0	243.8*	732.2#	65.4
Test Item	75.4	105.8	80.8	84.3
	90.4	95.7	82.3	91.5
	108.5	97.8	96.9	88.9
	130.2	116.5	93.8	87.9
	156.3	116.5	91.5	83.3
	187.5^P	133.8	115.4	88.5
	225^P	146.8	122.3	76.9
	270^P	214.4*	194.6#	67.7

^Pprecipitation/phase separation (oily droplets), are excluded from the evaluation

*RFI value of CD54 exceeded the positive criterion (CD54 ≥ 200%)

#RFI value of CD86 exceeded the positive criterion (CD86 ≥ 150%)

Results of the third h-CLAT run for the Test Item:

	Concentration (μg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control		100.0	100.0	100.0
DMSO Control		100.0	100.0	100.0
Positive Control (DNCB)	2.0	238.5*	271#	84.0
	3.0	272.4*	347.8#	79.3
Test Item	75.4	106.9	124.5	94.1
	90.4	108.1	127.9	93.0
	108.5	100.0	109.8	89.5
	130.2	113.4	145.6	102.4
	156.3	111.0	146.1	99.1
	187.5	121.1	130.4	92.7
	225^P	135.4	169.6#	87.0
	270^P	174.8	219.1#	77.8

^Pprecipitation/phase separation (oily droplets), are excluded from the evaluation

*RFI value of CD54 exceeded the positive criterion (CD54 ≥ 200%)

#RFI value of CD86 exceeded the positive criterion (CD86 ≥ 150%)

 

Applicant's summary and conclusion

Interpretation of results:

other: h-CLAT prediction: negative (no activation of dendritic cells) according to OECD 442E

Conclusions:

The test item with a log Pow of 2.7 did not activate THP-1cells up to a test item concentration of 156.3 μg/mL (limited by precipitation, oily droplets) under the test conditions of this study. The test item is therefore considered to be negative for the respective key event of the sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitizing potential of the test item dissolved or suspended in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The concentration of test item for the main experiment (h-CLAT) was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 312.5μg/mL test item up to the highest tested test item concentration (2500 μg/mL; maximum attainable concentration) in both XTT tests. In addition, the test item concentrations ≥ 312.5 μg/mL showed oily droplets. The mean CV75 value of both XTT tests was calculated as 222.85μg/mL.

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 75.4, 90.4, 108.5, 130.2, 156.3, 187.5, 225 and 270 μg/mL.

The test item with a log Pow of 2.7 was tested in 3 valid independent runs. The second run was repeated since the treatment time was not in the guideline recommended range.The RFI of CD54 was greater than the threshold of 200% in at least one concentration in 2 out of 3 independent runs. Furthermore, the RFI of CD86 was greater than the threshold of 150% in at least one concentration in all 3 independent runs. However, precipitations (oily droplets) were observed in the two highest (225 and 270 μg/mL; runs 1 and 3) and three highest (187.5, 225 and 270 μg/mL; run 2) test item concentrations, which were therefore excluded from the evaluation of the respective run.

Taking this in consideration, the RFI of CD86 and CD54 were slightly higher than 150% and 200%, respectively at 187,5 μg/mL in the first run.

As this slight increase was observed only in 1 out of 3 independent runs, the test item is considered negative in the h-CLAT.

In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥150% and CD54 ≥ 200%).

The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the relative cell viability was > 50%.

In conclusion, the test item with a log Pow of 2.7 did not activate THP-1cells up to a test item concentration of 156.3μg/mL (limited by precipitation, oily droplets) under the test conditions of this study.