Phenethyl cinnamate

Endpoint summary

Description of key information

The substance is not eye or skin irritating.

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:

08.08.2016-15.08.2016

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:

adopted July 28,
2015

Deviations:

yes

Remarks:

yes Instead of the MatTek EPI-200-SIT Kit, the MatTek EPI-200-PHO Test kit was used for the experimental performance. This Deviation had no influence on the outcome of the study (identical test kits with only the cell culture medium being different).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1st ATP 2009

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

6th revision, 2015

Deviations:

not specified

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: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Details on test system:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Surface: 0.6 cm.
- Shipment: with cool packs on medium-supplemented agarose gels.
- Pre-incubation: 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH) in the upper wells. Then transferred from upper wells into the lower wells for about 20.5 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg (~ 39 mg/cm2 according to guideline)

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

23.5h at 37+/-1.5°C, 5+/-1% CO2, 95+/-5% RH

Number of replicates:

3

Details on study design:

Details of the test procedure used:
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (Epi-200- PHO Kit, Lot No.:23349)
- Conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95%
- Washing: inserts gently rinsed with DPBS at least 15 times
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation with 0.3 mL of MTT solution for 3 hours at 37 ± 1 °C, 5 ± 0.5% CO2)

Data evaluation:
The following was calculated: The mean OD of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: Relative viability(%) = (mean OD test item / positive control / mean OD negative control) x 100. For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated.

Description of evaluation criteria:
For the current test, an irritation potential of the test item of H315, GHS Cat 2 according to UN GHS (published 2003, last (6th) revision 2015) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Historical data:
- Historical data positive control: Mean 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
- Historical data negative control: Mean Absorption: 1.74; Rel. Standard Deviation: 8.68%; Range of Absorbance: 1.48-1.98

Acceptability of the Assay:
The results are acceptable if (1) tissue viability is meeting the acceptance criterion, i.e. if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8 (negative control). If (2) the relative tissue viability of the positive control is ≤ 20% (positive control). If (3) the SD of 3 identical replicates is < 18%. (4) OD values should not be below historically established boundaries.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

102.6

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:

The acceptance criteria were met.

Results of the pre-experiment:

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour. Therefore, an additional test with viable tissues (without MTT addition) was not necessary.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.

Results after treatment with the test item and the controls:

Dose Group	Exposure Interval	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 Wells	Mean- Absorba nce of three wells blank corrected	Mean Absorbance of 3 tissues after blank correction	Rel. Absorbance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]**
Blank			0.038	0.039	0.039	0.038	0.000
Negative Control	60 min	Tissue 1	1.658	1.633	1.664	1.652	1.613	1.690	95.5	4.0	100.0
		Tissue 2	1.792	1.783	1.755	1.777	1.738		102.9
		Tissue 3	1.782	1.766	1.721	1.756	1.718		101.7
Positive Control	60 min	Tissue 1	0.104	0.100	0.101	0.101	0.063	0.060	3.7	5.3	3.5
		Tissue 2	0.094	0.096	0.096	0.095	0.057		3.4
		Tissue 3	0.097	0.098	0.097	0.097	0.059		3.5
Test Item	60 min	Tissue 1	1.806	1.764	1.730	1.767	1.728	1.734	102.3	4.1	102.6
		Tissue 2	1.912	1.813	1.810	1.845	1.807		106.9
		Tissue 3	1.720	1.700	1.693	1.704	1.666		98.6

* relative absorbance per tissue [rounded values]:100× (absorbance_tissue)/( mean absorbance_{negative control})

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

The mean relative absorbance value of the test item, corresponding to the cell viability, did not decrease (102.6%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Interpretation of results:

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

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test substance by means of the Human Skin Model Test. The test was performed according to OECD 439 and GLP.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes.

30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 μL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD≥0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item, the mean relative absorbance value was 102.6% 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 considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance is not 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:

12 Mar - 16 Mar 2001

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:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Jan 1997

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: Chbb:HM(SPF) — Littlerussian

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight of animals: 1.7 — 1.8 kg b.w.
- Housing: individually in PPO cages (floor area: 2576 sq.cm) with perforated floor.
- Diet: pelleted complete rabbit diet "Altromin 2123" from Altromin, D-32791 Lage, Lippe; ad libitum.
- Water: ad libitum; domestic quality drinking water acidified with hydrochloric acid to pH 2.5
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C
- Humidity: 55% ± 15%
- Air changes: 10 times/hour
- Photoperiod (hrs dark / hrs light): cycle of 12 hours light and 12 hours darkness. Light was on from 6 a.m. to 6 p.m.

DATES:
The experimental work was carried out between 12.03.2001 and 16.03.2001.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 g
- Concentration (if solution): undiluted

Duration of treatment / exposure:

Single dose, not rinsed until after the 24h examination.

Observation period (in vivo):

1, 24, 48 and 72 hours after treatment

Number of animals or in vitro replicates:

4

Details on study design:

TEST PROCEDURE
0.1g of the test article was placed in one eye of the rabbit, in the cup that formed by gently pulling the lower lid away from the eyeball.
The lids were then gently held together for 1 second.

OBSERVATIONS:
- Observations after 1h, 24h, 48h and 72h
- Additionnally, after the 24h reading, Fluorescein was instlled. The eye was rinsed with 20 mL 0.9% sodium chioride solution and examined again using UV light

SCORING SYSTEM:
See 'Any other information on materials and methods'

TOOL USED TO ASSESS SCORE: fluorescein-UV-light (after 24h).

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

Remarks:

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

Remarks:

not applicable

Irritation parameter:

chemosis score

Basis:

animal: #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal: #1, #3, #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility

Remarks:

not applicable

Irritation parameter:

conjunctivae score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal: #3, #4

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

other: reversibility

Remarks:

not applicable

Irritant / corrosive response data:

One hour after application of the test article animals No. 2085 and No. 2083 showed a diffuse, crimson red conjunctiva with individual vessels not easily discernbile, a swelling above normal and a discharge different from normal. Some conjunctival vessels definitely injected, a swelling above normal and a discharge different from normal were observed in animals No. 2084 and No. 2072.
24 hours after application of the test article animal No. 2085 showed some conjunctival vessels definitely injected. Some conjunctival vessels definitely injected and a swelling above normal were observed in animal No. 2083. Animals No. 2084 and No. 2072 were free of any signs of eye irritation.
48 and 72 hours after application of the test article all four animals, No. 2085, No. 2083, No. 2084 and No. 2072, 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 per kg	Parameters	Individual mean score*
2085/1.7	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.33
	conjunctiva chemosis	0.00
2083/1.8	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.33
	conjunctiva chemosis	0.33
2084/1.7	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.00
	conjunctiva chemosis	0.00
2072/1.7	cornea opacity	0.00
	iris	0.00
	conjunctiva redness	0.00
	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 item shall not be classified as eye irritating.

Executive summary:

In the current test the eye irritant effect of the test item was investigated according to the method recommended in the OECD Guideline No. 405 and EEC Guideline B.5. The study was performed in accordance with GLP.

Four female albino rabbits were exposed to 0.1 g of the test article in one 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 1, 24, 48 and 72 hours after dosing.

Slight to well-defined signs of irritation were observed on the treated eyes. However, 48 and 72 hours after application of the test article all four animals were free of any signs of eye irritation.

Based on these results, the substance was concluded to be not irritating to the eye.

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 three studies available assessing the skin irritant potential of the test substance to the skin.

 

The key study is an in vitro skin irritation study using the Human Skin Model Test (EpiDerm™). The study was performed according to OECD 439 and GLP. The substance was found to not reduce MTT nor to have an intensive colour, indicated that the interpretation of the test outcome would not be hampered. Each 3 tissues of the kit were exposed to 30 µL of the test item, the negative or the positive control for 60 minutes. The results obtained with the negative and positive control substances confirmed the quality of the tissues and the validity of the test system. After treatment with the test item, the mean relative absorbance value was 102.6% compared to the relative absorbance of the negative control. The threshold for irritancy is ≤ 50%. Hence, the test demonstrates that the test item is not to be considered as a skin irritant.

 

In the supporting studies (1975) the skin irritant effect of the test item was assessed during acute dermal toxicity tests in rabbits. In both studies a single application of 5000 mg/kg bw was applied to the skin and the animals were observed for 14 days. In the first test, no effects were observed in any of the 4 tested animals (Leberco Laboratories, 1975). In the second test, the test item was found to cause slight effects in 2 animals out of 10 (MB Research Laboratories, 1975).

These findings further demonstrate that the substance is not a skin irritant.

 

Eye irritation

 

There is one study available that assesses the possible irritation or corrosion potential of the test substance to the eye (2001).

The study was performed according to GLP and internationally accepted guidelines.

 

The eye irritating effect of the test item was investigated according to the OECD Guideline 405. Four female albino rabbits were exposed to 0.1g of the test article in one eye, while the other eye served as control. The eyes were examined and changes were graded according to a numerical scale 1, 24, 48 and 72 hours after dosing. Slight to well-defined signs of irritation were observed on the treated eyes. However, 48 and 72 hours after application of the test item, all four animals were free of any signs of eye irritation. According to the CLP regulation, the test item shall not be classified as eye irritating.

Justification for selection of skin irritation / corrosion endpoint: The study was well documented and according to GLP and internationally accepted guidelines. 

Justification for selection of eye irritation endpoint: The study was well documented and according to GLP and internationally accepted guidelines.

Justification for classification or non-classification

Skin irritation

The substance was found to be non-irritant following an in vitro Human Skin Model Test in accordance with OECD 439 and GLP.

Furthermore, observations from two acute dermal toxicity tests are also available. These in vivo test results cannot directly be used for classification purposes. However, they add to the conclusion that the substance should not be classified as irritant to the skin. The substance causes only slight effects in less than 30% of the animals.

Eye irritation

The CLP regulation states that a substance has irreversible effects on the eye (Category 1) if, when applied to the eye of an animal, a substance produces:

- at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days; and/ or

- at least in 2 of 3 tested animals, a positive response of: corneal opacity >= 3 and/or iritis > 1,5

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

The substance is considered irritating to eyes (Category 2) if, when applied to the eye of an animal, a substance produces:

- at least in 2 of 3 tested animals, a positive response of: corneal opacity >= 1 and/or
 iritis >= 1, and/or
 conjunctival redness >= 2 and/or
conjunctival oedema (chemosis) >= 2

- calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

According to these criteria of the CLP regulation the test item should not be classified as an eye irritant.