Benzyl cinnamate

Endpoint summary

Description of key information

The substance was found not to be skin or eye irritating according to the CLP criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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.

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 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

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.

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)

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04.10.1999 - 08.10.1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Feb. 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Guideline B.5 "Acute Toxicity (Eye Irritation)"

Version / remarks:

Jan. 1997

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight: 2.6 - 3.0 kg b.w.
- Housing: individually in PPO cages (floor area: 2576 cm2) with performed floor.
- Diet: pelleted complete rabbit diet "Altromin 2123" from Altromin, D-32791 Lage, Lippe; ad libitum.
- Water: free access to domestic drinking water acidified with hydrochloric acid to pH 2.5
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C
- Humidity: 55% ± 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): cycle of 12 hours light (06 to 18h) and 12 hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

Single exposure without rinsing.

Observation period (in vivo):

1, 24, 48 and 72 hours after treatment

Number of animals or in vitro replicates:

4 females

Details on study design:

TESTING PROCEDURE
- The day before testing both eyes of the animals were examined with a hand held inspection lamp fitted with white and UV-light and magnifying glass with 2 x magnification to ensure there were no defects or irritation. The examination was performed before and after instillation of Fluorescein.

- The left eye was treated. The right eye remained untreated and served as control.
- 0.1 ml of the warmed test item was placed in the left eye of the rabbits by gently pulling the lower lid away from the eyeball to form a cup into which the test article was dropped. The lids were then gently held together for 1 second.

- The eyes were examined and the grade of ocular reaction was recorded one hour and 24 hours later. After the first 24 hour reading Fluorescein was instilled. After rinsing with 20 ml 0.9% sodium chloride solution the eyes were examined again using UV-light to detect possible comeal damage.
- The eyes were also examined 48 and 72 hours after the treatment.

EVALUATION OF THE REACTIONS (see tables below)

ASSESSMENT
A substance or a preparation is considered to be irritant if it causes ocular lesions which occur within 72 hours after exposure and which persist for at least 24 hours.

Ocular lesions are significant if the rnean scores of the eye irritation test have any of the following values:
- cornea opacity ≥ 2 but < 3
- iris lesion ≥ 1 but < 1.5
- redness of conjunctivae ≥ 2.5
- oedema of conjunctivae (chemosis) ≥ 2
or, in the case where the test has being completed using three animals if the lesions, on two or more animals, are equivalent to any of the above values except that for iris lesion the value should be equal or greater than 1 but less than 2 and for redness of the conjunctivae the value should be equal or greater than 2.5.
In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be used in calculating the respective mean values.

A substance or a preparation is considered to be able to cause serious damage to eyes if it causes severe ocular lesions which occur within 72 hours after exposure and which persist for at least 24 hours.

Ocular lesions are severe if the means of the scores of the eye Irritation test have any of the values:
- cornea opacity ≥3
- iris lesion ≥ 1.5
The same shall be the case where the test has been completed using three animals if these lesions, on two or more animals, have any of the values:
- cornea opacity ≥ 3
- iris lesion = 2
In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be used in calculating the respective mean values.

Ocular lesions are also severe when they are still present at the end of the observation time.

Ocular lesions are also severe if the substance or preparation causes irreversible colouration of the eyes.

Irritation parameter:

cornea opacity score

Basis:

animal: #1, #2, #3, #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Irritation parameter:

iris score

Basis:

animal: #1, #2, #3, #4

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: reversibility: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal: #1, #2, #3, #4

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal: #1, #2, #3, #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility: not applicable

Irritant / corrosive response data:

One hour after application of the test article animals No. 1565, No. 1571, No. 1572 and No. 1573 showed some conjunctival vessels definitely injected.
After 24 hours some conjunctival vessels were definitely injected in all animals. After 48 and 72 hours all animals were free of any signs of eye irritation.

Scores for ocular lesions:

*individual mean score: Only the scores from the readings after 24, 48 and 72 hours are included in the calculation of the individual mean scores.

Rabbit No/Weight (kg)	Parameters	Individual mean score*
1565/2.9	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.33
	conjunctiva chemosis	0.00
1571/2.6	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.33
	conjunctiva chemosis	0.00
1572/2.8	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.33
	conjunctiva chemosis	0.00
1573/3.0	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.33
	conjunctiva chemosis	0.00

Interpretation of results:

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

Conclusions:

The test substance shall not be classified as eye irritating.

Executive summary:

In the current study the eye irritant effect of the test item was investigated according to the method recommended in the OECD Guideline 405 and EEC Guideline B.5.

Four female albino rabbits were exposed to 0.1 mL of the undiluted test articie in the left eye, while the other eye remained untreated and served as control. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing.

Slight signs of irritation were observed on the treated eyes, however, these were reversible and according to the criteria set out in the CLP regulation the test item shall not be classified as eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

There are 3 studies available that assess the possible irritation potential of the test substance to the skin. The study from 2016 was performed according to GLP and internationally accepted guidelines, the supporting studies were not in conformance to any guideline nor to GLP. The key study is used for classification, the supporting studies could not be used for classification because they lack e.g. individual animal results. The supporting studies are studies designed for other endpoints as well, however, the information was added here because of their relevance.

In the key 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 item did not reduce MTT, change the colour or have intensive, intrinsic colour and so no additional tests with freeze-killed or viable tissues were necessary.

The human skin model EpiDermTM was treated with the test item, the negative (DPBS) or the positive control (5% SLS) for 60 minutes. The acceptance criteria of the test were met.

The test item showed a mean relative absorbance value of 86.0% compared to the negative control. This value is above the threshold for irritancy of ≤ 50% and thus the test item is not considered to possess irritant potential.

In the first supporting study (1976) the skin irritation effects were assessed as part of an OET test on guinea pigs. Only a mean scoring value of 6 -8 animals after 1 day of treatment is presented in the study and some slight irritation after a single application of the test item was observed, however, this was not again assessed at 48 and 72 hours.

In the second supporting study (1977) the skin irritation of test substance was assessed in guinea pigs as part of an OET test and the lowest concentration to produce mild redness was determined to be 3% after 1 application (minimal irritating concentration).

Taken together, the test item shall not be classified as skin irritating.

Eye irritation

There is only one study available assessing the eye irritation potential of the test substance and it was performed according to GLP and internationally accepted guidelines.

 

The study was according to the OECD Guideline No. 405 and 4 female albino rabbits were exposed to 0.1 mL of the test article in the left eye, while the other eye remained untreated and served as control. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing.

Slight signs of irritation were observed on the treated eyes. However, according to the CLP criteria, the test substance shall not be classified as eye irritating.

Justification for classification or non-classification

Skin irritation

In a recent in vitro skin irritation study the test item showed a mean relative tissue viability of 86.0%.

The test item is considered irritant to the skin if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Thus, based on the available data on skin irritation, the test item does not meet the criteria for classification according to Regulation (EC) 1272/2008 (CLP).

Eye irritation

The observed effects of conjunctival redness are slight, with a mean (24 - 48 - 72h) score of 0.33 in all tested animals. As this value is below 2, and the observed effects are fully reversible, classification of the test substance is not required according to the criteria set out in EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).