Benzyl cinnamate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.06 - 20.06.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

yes

Remarks:

However, the deviations did not influence the outcome or the validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium fuer Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl cinnamate

EC Number:

203-109-3

EC Name:

Benzyl cinnamate

Cas Number:

103-41-3

Molecular formula:

C16H14O2

IUPAC Name:

benzyl 3-phenylacrylate

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not applicable

Source strain:

other: not applicable

Justification for test system used:

Dermal irritation is usually determined in vivo in the Draize rabbit skin irritation test as described in OECD guideline 404. Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDermTM and EpiSkinTM and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM tissues
- Tissue batch number(s): 23341

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5°C, 5 ± 0.5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5°C, 5 ± 0.5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT solution: 300 μL
- Incubation time: 42 hours at 37 ± 1°C, 5 ± 0.5% CO2
- Microplate reader: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Measurement: mean values from the 3 wells per tissue were calculated.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Positive control
- Viability: 4.64%
- Rel. Standard Deviation: 11.2%
- Range of Viabilities: 4.00% - 5.90%
- Mean Absorption: 0.0803
- Rel. Standard Deviation: 12.6%
- Range of Absorbance: 0.066 - 0.097
Negative control
- Mean Absorption: 1.74
- Rel. Standard Deviation: 8.68%
- Range of Absorbance: 1.48 – 1.98
Data of 31 studies performed from July 2015 until March 2016

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test item is considered Category 1 or Category 2 (UN GHS published 2003, last (6th) revision 2015) if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

ACCEPTANCE CRITERIA
1. Negative control: The absolute OD 570 nm of the negative control tissues / tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.
2. Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
3. Standard deviation: The SD of 3 identical replicates should be < 18%. OD values should not be below historically established boundaries.
Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system or rather of the test kit.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

undiluted

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42.75 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Prior to use, the test item was melted at 37±2°C for 25 minutes in a water bath and was, subsequently, treated as liquid. The test item passed the MTT- and the colour interference pre-tests.
Treatment with the negative control was well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 4.5% thus ensuring the validity of the test system.
The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 11%, ensuring the validity of the study.

Any other information on results incl. tables

Results after treatment with the test item and the controls

Dose Group	Tissue	Absorbance 570 nm Well 1		Absorbance 570 nm Well 2		Absorbance 570 nm Well 3	Mean absorbance of 3 Wells	Mean absorbance of 3 Wells blank corrected	Mean absorbance of 3 tissues after blank correction*	Rel. Absorbance (%) Tissue 1,2 and 3*	Realtive Standard Deviation (%)	Mean Rel. Absorbance (% of Negative Control)**
Blank		0.037		0.038		0.038	0.038	0.000
NK	1	1.870		1.865		1.837	1.857	1.820	1.889	96.3	3.3	100
	2	1.954		1.947		1.944	1.948	1.911		101.1
	3	1.997		1.966		1.963	1.975	1.938		102.6
PC	1	0.123		0.127		0.127	0.126	0.088	0.086	4.7	6.5	4.5
	2	0.131		0.126		0.126	0.128	0.090		4.8
	3	0.118		0.117		0.117	0.117	0.079		4.2
TM	1	1.823		1.804		1.818	1.818	1.780	1.626	88.4	10.6	86
	2	1.483		1.467		1.477	1.477	1.440		94.2
	3	1.707		1.687		1.694	1.694	1.657		76.2

* relative absorbance per tissue [rounded values]: 100*(absorbance tissue) / (mean absorbance negative control)

** relative absorbance per treatment group [rounded values]: 100*(mean absorbance test item/positive control) / (mean absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

Under the experimental conditions reported, the test item is non irritant to skin.

Executive summary:

In the current study the irritation potential of the test item was assessed in an in vitro study by means of the Human Skin Model Test according to OECD TG 439 and GLP.

The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls is used to predict the skin irritation potential and is used for the purpose of classification as irritating or non-irritating. The test chemical is regarded as skin irritant (UN GHS and EU CLP) if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%. However, a limitation of this test method is the unability to distinguish between Category 1 (skin corrosive) and Category 2 (skin irritant). Thus, a positive result in this assay requires further testing.

The test item did not reduce MTT in the direct MTT reduction pre-test, and did not change the colour in the colour interference pre-test. Also, its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Tissues of the human skin model EpiDermTM were treated with the test item, the negative (DPBS) or the positive control (5% SLS) for 60 minutes. The test item, the negative control or the positive control were spread to match the surface of the tissue.

The absorbance values after treatment with the negative control were well within the required range of acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8, assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance compared to the negative control, assuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 86.0% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not to be considered to possess irritant potential.

In conclusion, under the experimental conditions reported, the test item is non irritant to skin.