trans-cinnamic acid

Endpoint summary

Description of key information

A weight of evidence approach is applied for the skin sensitisation endpoint. The dataset considered consists of the following:

- Literature review (not reported in the dossier, since there was no available data).

- (Q)SAR assessment

- OECD 442C

- OECD 442D

- OECD 442E

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16-19 October 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:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor / batch: 74271, lot: 20171106
- Expiration date of the lot/batch: 10 April 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15° to 25°C) dry, in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Final dilution of a stock liquid: 100mM solution in acetonitrole

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Test system: Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA-OH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA-OH). The molecular weight is 751 g/mol for SPCC and 776 g/mol for SPCL.
Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: AnaSpec
Storage: Frozen (-10°C to -30°C)

Positive control results:

Cysteine:
Standard linearity: r2>0.999, positive control depletion:72.1% (SD, 0.05%, n=3)
Lysine:
Standard linearity: r2>0.999, positive control depletion: 51.8% (SD, 0.67%, n=3)

Run / experiment:

other: mean value

Parameter:

other: mean SPCC depletion (%)

Value:

-1.15

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: mean value

Parameter:

other: mean SPCL depletion (%)

Value:

-0.645

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Peptide calibration graphs and typical sample chromatograms were included in the test report.

All analytical acceptance criteria for each peptide run were met:

		Peptide		Standard Linearity		Positive control depletion (%)		Reference controls		Test item
Acceptance criteria		Cysteine		r^2>0.99		60.8-100 (SD <14.9%)		0.45-0.55 mM (CV <15%)		SD <14.9%
		Lysine		r^2>0.99		40.2-69.0 (SD <11.6%)		0.45-0.55 mM (CV <15%)		SD <11.6%
Achieved results		Cysteine		r^2>0.999		72.1 (SD, 0.05%, n=3)		B: 0.494 mM (CV 0.52%, n=6)		SD 1.03% (n=3)
		Lysine		r^2>0.999		51.8 (SD, 0.67%, n=3)		B: 0.504 mM (CV 0.25%, n=6)		SD 0.26% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

The depletion of peptide in the presence of trans-cinnamic acid was:

	Mean peak area of peptide	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by Trans-Cinnamic acid (%)
Cysteine	Control B: 798210 (n=6)	807360 (n=3)	-1.15
Lysine	Control B: 806760 (n=6)	811960 (n=3)	-0.645

Applying the following depletion model (below), reactivity is classed as “no to minimal” and the DPRA prediction is therefore negative and trans-cinnamic acid is therefore predicted not to be a potential skin sensitizer. 

Mean of cysteine and lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

There were no co-elution peaks in either the Cysteine or Lysine assays.   

Interpretation of results:

study cannot be used for classification

Conclusions:

The reactivity of trans-cinnamic acid is classed as “no to minimal” and the DPRA prediction is therefore negative. Trans-cinnamic acid is therefore predicted not to be a potential skin sensitiser.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of trans-cinnamic acid. 

Solutions of trans-cinnamic acidwere successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With no depletion of either the Cysteine or Lysine peptides in the presence of trans-cinnamic acid, this places the test item in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitiser. 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 February - 28 February 2019

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:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The study is performed as part of a weight of evidence approach to skin sensitisation testing that incorporates in silico, in vitro and in chemico methods.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / batch: 74271 / Lot: 20171106
- Expiration date of the lot/batch: 10 April 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (10 - 30˚C), desiccated, in the dark
- Stability under test conditions: stable
- Solubility: The test item, Trans-cinnamic acid, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

The test was conducted in accordance with the following test guideline and protocol:
OECD Guideline for the Testing of Chemicals, In Vitro Skin Sensitisation Assays Addressing The Aop Key Event On Keratinocyte Activation, Guideline 442D, June 2018.
DB-ALM Protocol no 155: KeratinoSens™, March 2018.

Cell culture: transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland).
The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10270) and 5.5 mL Geneticin (Gibco 10131).

Storage: Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196C under liquid nitrogen, in DMEM containing 10% dimethyl sulphoxide and 20% FBS, were thawed rapidly at 37°C in a water-bath.
Cell preparation: The cells were then resuspended in a total of 10 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.

Cell passage: Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks were pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages from frozen stock.

Preparation of test cell cultures: The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. One well of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Positive control: Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020)

Preparation of test item: A stock solution of the test item, Trans-cinnamic acid, was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO.

number of tests required: Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.

PARAMETERS EVALUATED
The following parameters were calculated in the KeratinoSens™ test method:
• the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
• the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained; and
• the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability

Positive control results:

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde, were 9.34 μM and 9.11 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory (see Table 5). The average induction in the three replicates for cinnamic aldehyde at 64 µM were 3.79 and 3.62 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.
The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 13.7% and 13.0% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Key result

Run / experiment:

other: Test 1

Parameter:

other: Imax

Value:

1.24

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Test 2

Parameter:

other: Imax

Value:

1.03

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The Imax for both tests was <1.5 fold compared to the DMSO control and therefore the EC1.5 could not be calculated. The cellular viability did not fall below 107.98% in test 1 and 90.60% in test 2 and therefore the IC30 and IC50 could not be calculated.

There was no response for luciferase induction.

Interpretation of results:

study cannot be used for classification

Conclusions:

It was concluded that Trans-cinnamic acid gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that Trans-cinnamic acid is not a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

in Silico

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

An assessment of the sensitising potential of trans-cinnamic acid was made using a number of QSAR programs. The assessment results and QMRF for each program is presented in the attached report.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 November - 30 November 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 sensitisation, h-CLAT)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The study is performed as part of a weight of evidence assessment for skin sensitisation in line with the REACH requirements.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor / Lot: 20171106, Batch: 74271
- Expiration date of the lot/batch: 10 April 2020
- Purity test date: 09/04/2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, under moisture protection
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not indicated by Sponsor
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated by Sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Final preparation of a solid: prepared in culture medium which formed a stable suspension/dispersion.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Suspension / dispersion

Details on the study design:

Controls for Cytotoxicity Test and h-CLAT
Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously.

Medium Control and Solvent Control for the Test Item
Name: Culture medium

Positive Control (h-CLAT)
Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 2 and
3 µg/mL, Purity ≥ 99%)
Solvent: DMSO

Solvent Control for the Positive Control (h-CLAT)
Name: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2%, Purity ≥ 99%

TEST ITEM PREPARATION
The maximum concentration of test item was 5000 µg/mL in culture medium, as tested by a solubility test.
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilution. To prepare the test item, sonication of the formulation for 5 minutes was performed.

Experimental Design and Procedures of h-CLAT

The test item was tested in two independent runs. The tests were performed on different days. The test item was prepared separately for each run.

Treatment of the Cells
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2-8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2-8 °C) with 1 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

The following concentrations of the test item were tested in the main experiments (h-CLAT):
249, 298, 358, 430, 516, 619, 743 and 891µg/mL

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%, with one exception. The CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first and second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this exception is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in both h CLAT runs exceeded the positive criteria.

Run / experiment:

other: Run 1

Parameter:

other: Number of valid test item dose levels exceeding positive criteria based of RFI for CD54 antibody

Remarks:

(>200%)

Value:

2

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Run 2

Parameter:

other: Number of valid test item dose levels exceeding positive criteria based of RFI for CD54 antibody

Remarks:

(>200%)

Value:

3

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Run 1

Parameter:

other: Number of valid test item dose levels exceeding positive criteria based of RFI for CD86 antibody

Remarks:

(>150%)

Value:

2

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Run 2

Parameter:

other: Number of valid test item dose levels exceeding positive criteria based of RFI for CD86 antibody

Remarks:

(>150%)

Value:

3

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 µg/mL up to the highest tested concentration (5000 µg/mL) in both cytotoxicity tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both cytotoxicity tests was calculated as 742.45 µg/mL.


DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated. Details were not provided in the study report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
- Acceptance criteria met for positive control: yes

 Results of the Dose Finding Assay (Cytotoxicity Test)

Results of the first Cytotoxicity Test for the Test Item Trans-Cinnamic Acid

 

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	98.09
Test Item	39.1	no	96.94
	78.1	no	97.71
	156	no	97.84
	313	no	98.88
	625	no	95.29
	1250	no	31.02
	2500	yes	1.98
	5000	yes	0.46

Shaded teste groups: cytotoxic effects occurred by the Flow Cytometric Evaluation (< 75% cell viability)

The CV75 value of the first Cytotoxicity Test: 777.88 µg/m

 

Results of the second Cytotoxicity Test for the Test Item Trans-Cinnamic Acid

 

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	96.83
Test Item	39.1	no	96.93
	78.1	no	96.9
	156	no	96.57
	313	no	95.85
	625	no	88.34
	1250	no	13.35
	2500	yes	15.21
	5000	yes	0.13

Shaded test groups: cytotoxic effects occurred by the Flow Cytometric Evaluation (< 75% cell viability)

The CV75 value of the second Cytotoxicity Test: 707.02 µg/mL

The mean CV75 value of both Cytotoxicity Tests: 742.45 µg/mL

 Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item Trans-Cinnamic Acid

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	96.69
DMSO Control	-	100.0	100.0	97.35
Positive Control (DNCB)	2.0	89.2^#	255.2*	93.50
	3.0	305.4*	332.4*	90.26
Test Item	249	165.0	51.0	92.60
	298	165.8	58.4	92.61
	358	180.3	361.5*	91.18
	430	200.9*	68.1	89.28
	516	180.3	297.5*	83.92
	619	94.0	51.0	76.93
	743	219.7*	5.5	69.53
	891	21.4	-139.1	44.54

Shaded test groups: cell viability below 50%, are excluded from the evaluation

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).

^#    CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%)

 

Results of the second h-CLAT run for the Test Item Trans-Cinnamic Acid

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	96.45
DMSO Control	-	100.0	100.0	95.50
Positive Control (DNCB)	2.0	167.0^#	237.6*	87.32
	3.0	297.8*	229.1*	85.23
Test Item	249	124.5	59.8	94.63
	298	137.7	86.0	93.87
	358	134.0	75.4	93.87
	430	192.5	101.9	92.97
	516	356.6*	72.0	82.46
	619	392.5*	154.7*	61.75
	743	254.7*	170.1*	58.08
	891	354.7*	268.6*	49.92

Shaded test groups: cell viability below 50%, are excluded from the evaluation

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

^#    CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item Trans-Cinnamic Acid with an estimated log Pow of 2.1 activated THP-1 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 (AOP).

Executive summary:

 Summary

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of Trans-Cinnamic Acid prepared inculture medium which formed a stable suspension/dispersion when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of Trans-Cinnamic Acidwas previously determined by two cytotoxicity tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 µg/mL up to the highest tested concentration (5000 µg/mL) in both cytotoxicity tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both cytotoxicity tests was calculated as 742.45 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT): 249, 298, 358, 430, 516, 619, 743 and 891µg/mL

The test item with an estimated log Pow of app. 2.1 (estimated via KOWWIN v1.68) and 2.13 (Experimental Database Structure Match) was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both independent runs. No dose response could be observed in the first h-CLAT run, but a slight dose response could be observed for CD54 and CD86 in the second h-CLAT run. The h-CLAT prediction is considered positive for the test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in both h-CLAT runs did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in both h-CLAT runs exceeded the positive criterion.

In conclusion, the test item Trans-Cinnamic Acid with an estimated log Pow of app. 2.1 (estimated via KOWWIN v1.68) and 2.13 (Experimental Database Structure Match) activated THP-1 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 (AOP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The weight of evidence assessment indicates that the substance is not classified as a skin sensitiser in accordance with Regulation (EC) No . 1272/2008 (EU CLP). No further testing is considered to be necessary.