Phenethyl cinnamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.04.-03.05.2017

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:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

22 Jul. 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Phenethyl cinnamate

EC Number:

203-120-3

EC Name:

Phenethyl cinnamate

Cas Number:

103-53-7

Molecular formula:

C17H16O2

IUPAC Name:

phenethyl cinnamate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Animal Husbandry:
- Type & size of cage: Polysulfone cage, 200W-320D-140H (mm)
- Bedding: Teklad sani-chips
- Number of animals per cage: 2–5 animals/cage (during the quarantine-acclimation period) / 2–3 animals/cage (during the study)
- Temperature Measurement value:
20.9–22.9°C (Dose range finding study)
21.2–23.0°C (Main study)
permissible range: 19.0–25.0°C
- Relative humidity Measurement value:
39.1–52.6% (Dose range finding study)
44.9–53.0% (Main study)
permissible range: 30.0–70.0%
- Air changes 10–15 clean, fresh, filtered air changes per hour
- Lighting: 12 hour light/dark cycle (7 AM to 7 PM via automated timer)
- Intensity of illumination: 150–300 Lux

Feed:
- Type: Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, Lot No. 2918C-120516MA, Supplier Envigo RMS, Inc., U.S.A.)
The feed was placed in feeders and provided ad libitum.

Drinking Water:
- Type and method of water supply: public tap water in Cheongju-si was filtered and irradiated by ultraviolet light and provided ad libitum in a quarantine room and by an automatic watering system in an animal room.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

- Dose range finding study
Dose volume 25 μL/ear
Negative control 0 0 (v/v), test substance 2.5 (w/v), 5 (w/v), 10 (w/v), 25 (w/v) and 50 (w/v)

- Main study
Dose volume 25 μL/ear
Negative control 0 0 (v/v), test substance 10 (w/v), 25 (w/v), 50 (w/v), positive substance 25 (v/v)

No. of animals per dose:

- dose range finding study: 2 females per group
- main study: 5 females per group

Details on study design:

Dosing:
- Route
Topical application to the dorsum of each ear
- Justification for the route of administration
The topical route was chosen to assess the skin sensitization by topical exposure of the test substance.
- Method of administration
A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. The dose level of the positive substance was selected at 25%, which is the dose recommended in the guideline. Negative control animals were dosed with the vehicle, AOO solution.

Dose Range Finding Study
A dose range finding was conducted under Non-GLP conditions.
- Dose levels and administration
Dose selection was based on the consecutive doses and dose levels were selected from a series of appropriate concentrations such as 50, 25, 10, 5 and 2.5%.
- Study method
The dose range finding study was conducted identical to the method of the main study, but lymph node proliferation measurement was not performed.
- Justification for selection of dose levels in the main study
Information on toxicity was assessed by confirming systemic toxicity and local irritation at the site of administration in the dose range finding study. A loss of body weight by 5% or more compared to pre-dose was judged to be due to systemic toxicity. An erythema score of ≥ 3 or ear thickness of ≥ 25% compared to pre-dose was judged to be due to excessive local irritation.
As a result of the dose range finding study, toxicity was not confirmed in high dosed (50%) animals, thus 50% was selected as the high dose of the test substance for the main study and two additional lower dose levels (25 and 10%) were established.
In addition, the positive (25% HCA) and negative control (Vehicle) groups were included in the main study.

Main Study:
- Dose levels and administration
Dose selection was based on the preliminary dose range finding study.
- Clinical signs
All animals were observed at least once daily for 6 days for mortality, general condition, clinical signs (changes in nervous system function: e.g. pilo-erection, ataxia, tremors and convulsions, changes in behavior: e.g. aggressiveness, change in grooming activity, marked change in activity level; changes in respiratory patterns: i.e. changes in frequency and intensity of breathing such as dyspnea, gasping and rale, and changes in food and water consumptions), or such as local irritation at the site of application.
- Body weights
Body weights were recorded prior to dosing (Day 1) and prior to necropsy (Day 6).

Skin Sensitization Evaluated:
- Erythema score
Both ears of each mouse were observed for erythema and scored for 6 days according to the erythema score.
- Ear thickness
Ear thickness (historical data: 0.19-0.20 mm) measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after first treatment) and Day 6 (the day of necropsy). On Day 6, ear thickness was determined by ear punch weight determinations.
- 5-Bromo-2-deoxyuridine (BrdU) injection
A volume of 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected inter-peritoneally on Day 5.
- Necropsy
Approximately 24 hours (24 h) after BrdU injection, the animals were euthanized under CO2 gas. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal.
- Preparation of cell suspension
For each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared using a #70 nylon mesh to generate a single cell suspension. In each case, the target volume of the LNC suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1–0.2 in the NC group.
- Determination of cellular proliferation
BrdU was measured by ELISA using a commercial kit. Briefly, 100 µL of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. Absorbance at 370 nm (Emission wavelength, em) with a reference wavelength of 492 nm (reference wavelength, ref) was measured.
- Results of absorbance of each well (absorption, ABS) were used to calculate the BrdU labeling index by substituting into the following equation:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
- The mean BrdU labelling index of each group was substituted into the following equation of stimulation index (stimulation index, SI) to calculate SI.
SI = Mean of BrdU labeling index for the test substance group / Mean of BrdU labeling index for the negative control group

Acceptance Criteria:
Evaluation of the validity of the study results was performed based on the following criteria:
Positive Control: Result:
α-hexylcinnamaldehyde SI ≥ 1.6

Evaluation Criteria:
SI: Result:
SI < 1.6 Negative
SI ≥ 1.6 Positive

Evaluation:
The EC1.6 value was calculated and reported. Since at least one concentration showed SI of <1.6, the following formula was employed: EC1.6 =c + [(1.6-d) / (b-d)] x (a-c).
a = dose concentration with higher SI
b = higher SI value
c = dose concentration with lower SI
d = lower SI value

The EC1.6 value is used to classify the test substance as follows:
EC1.6 Value (%): ECETOC Potency Classification:
≥10 to ≤100 Weak
≥1 to ≤10 Moderate
≥0.1 to ≤1 Strong
<0.1 Extreme

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index.

Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between thenegative control group (G1) and each of the test substance groups (G2–G4) or positive substance group (G5). Since it was not significant, Kruskal-wallis test was employedon heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group (G1) and each of the test substance groups (G2–G4) or positive substance group (G5).

Kruskal-wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group (G1) and each of the test substance groups (G2–G4) or positive substance group (G5).

Results and discussion

Positive control results:

The positive control confirmed the validity of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

negative control

Results 2

Key result

Parameter:

SI

Value:

ca. 0.8

Test group / Remarks:

substance groups at 10%

Results 3

Key result

Parameter:

SI

Value:

ca. 0.95

Test group / Remarks:

substance groups at 25%

Results 4

Key result

Parameter:

SI

Value:

ca. 1.01

Test group / Remarks:

substance groups at 50%

Results 5

Key result

Parameter:

SI

Value:

ca. 1.68

Test group / Remarks:

positive control group at 25%

Any other information on results incl. tables

Clinical Signs

There were no abnormal clinical signs or deaths in any dosing group during the observation period.

Body Weights

In the negative control group, the mean body weights were 19.3 and 20.0 g on Day 1 and Day 6 after dosing, respectively.

In the test substance groups at 10, 25 and 50%, the mean body weights (Day 1 and Day 6) were 19.2, 20.3 and 18.9 as well as 19.2, 19.3 and 19.9 g, respectively. There were no significant differences when compared to the negative control group (19.3 and 20.0 g).

In the positive control group at 25%, the mean body weights for Day 1 and Day 6 were 18.7 and 19.4 g, respectively. There were no significant differences when compared to the negative control group.

Erythema Scores

In the negative control group, the mean erythema score was 0–0 from Day 1 to Day 6 after dosing.

In the test substance groups at 10, 25 and 50%, the mean erythema scores were all 0–0 from Day 1 to Day 6. There were no significant differences when compared to the negative control group.

In the positive control group at 25%, the mean erythema score was in the range of 0–1 from Day 1 to Day 6. The effects observed were significantly increased when compared to the negative control group (p<0.05: Days 3 and 4, p<0.01: Days 5 and 6).

Ear Thickness

In the negative control group, the mean ear thickness was 0.19–0.19 mm from Day 1 to Day 6 after dosing.

In the test substance groups at 10, 25 and 50%, the mean ear thickness values (Day 1 to Day 6) were 0.19–0.19, 0.19–0.19 as well as 0.19–0.19 mm, respectively. There were no significant differences when compared to the negative control group.

In the positive control group at 25%, the mean ear thickness values were 0.18–0.21 mm on Day 1 to Day 6, respectively. The effects observed were significantly increased when compared to the negative control group (p<0.01: Day 6).

Ear Weights

In the negative control group, the mean ear weight was 12.4 mg.

In the test substance groups at 10, 25 and 50%, the mean ear weights were 13.7, 12.4 and 12.8 mg, respectively. There was a significant increase detected when compared to the negative control group (p<0.01: 10%). The differences were judged not to be of biological relevance since variation was within historical control value and no dose dependency was detected. Therefore, the erythema score and ear thickness were confirmed as normal.

In the positive control group at 25%, the mean ear weight was 14.0 mg. There was a significant increase when compared to the negative control group (p<0.01).

 

Stimulation Index

The mean stimulation index of the negative control group was 1.00.

In the test substance groups at 10, 25 and 50%, mean stimulation indices of 0.80, 0.95 and 1.01 were observed, respectively. There were no significant differences detected when compared to the negative control group.

In the positive control group at 25%, the mean stimulation index was 1.68. There was a significant increase when compared to the negative control group (p<0.01).

Mean Stimulation Index

Group/		BrdU labelling index	Stimulation index
Dose (%)
G1	Mean	0.15	1
0	S.D.	0.05	0.34
	N	5	5
G2	Mean	0.12	0.8
10	S.D.	0.02	0.15
	N	5	5
G3	Mean	0.14	0.95
25	S.D.	0.04	0.25
	N	5	5
G4	Mean	0.15	1.01
50	S.D.	0.03	0.22
	N	5	5
G5	Mean	0.25	1.68
25	S.D.	0.05	0.35
	N	5	5
			^**

G1: Acetone: olive oil (4:1 v/v), G2-G4: Test substance, G5: α-hexylcinnamaldehyde 

S.D.: Standard deviation

N: Number of animals

^**p<0.01, Significant difference from the negative control group (G1) by Dunnett's t-test.

Applicant's summary and conclusion

Interpretation of results:

other: no evidence of skin sensitization

Conclusions:

In conclusion, the test substance did not show any evidence of skin sensitization on CBA/N mice under the conditions of this study. The positive control confirmed the validity of the test system.

Executive summary:

The purpose of this study was to assess the skin sensitization potential of the test substance after application to the dorsum of each ear of female CBA/N mice. The study was performed according to OECD guideline 442B and in compliance to GLP.

The dose range finding study was conducted at dose levels of 2.5, 5, 10, 25 and 50% to determine the high dose level for the main study. In the dose range finding study, clinical signs, body weights, erythema score, ear thickness and ear weights by the toxicity of the substance was evaluated.

Based on the results of the dose range finding study, the high dose level for the main study was selected as 50%.Two additional lower dose levels (25 and 10%) were applied.

As a result of the study, no abnormal clinical signs or deaths were observed in any test substance group during the observation period.

In the test substance groups, the ear weight was significantly different when compared to the negative control group only at the lowest test concentration of 10%. The differences were judged not to be biologically relevant. The erythema score, ear thickness, body weight, and stimulation index (SI) were not significantly different when compared to the negative control group. The stimulation index (SI), which is an index of skin sensitization, was calculated to be <1.6 in all groups.

In the positive control group at 25 %, the erythema score, ear thickness, ear weight and stimulation index were significantly increased when compared to the negative control group, and the stimulation index (SI) was calculated to be 1.68, which is over 1.6.

Based on the result of this study (Local lymph node assay: BrdU- ELISA), the test substance did not show any evidence of skin sensitization on CBA/N mice under the conditions of this study.