Phenethyl benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

The study was done in 2011, before the changes in legislation concerning animal testing entered into force.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2011 - December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

data is from experimental reports

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

24 April 2022

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2004/73/EC L 152, 2004

Deviations:

not specified

Principles of method if other than guideline:

Mouse Local Lymphnode Assay was conducted to determine the allergenic potential of the test chemical

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Phenethyl benzoate

EC Number:

202-336-5

EC Name:

Phenethyl benzoate

Cas Number:

94-47-3

Molecular formula:

C15H14O2

IUPAC Name:

2-phenylethyl benzoate

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): 2-phenylethyl benzoate
- Molecular formula: C15H14O2
- Molecular weight: 226.274 g/mol
- Substance type: organic
- Physical state: Liquid
- Smiles notation: c1(C(OCCc2ccccc2)=O)ccccc1
- InChl: 1S/C15H14O2/c16-15(14-9-5-2-6-10-14)17-12-11-13-7-3-1-4-8-13/h1-10H,11-12H2

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Nulliparous and non-pregnant female mice were used in the study.
Age Range: 8-9 weeks at start of dosing
Body Weight Range: 19-26 grams at the outset (Day 1) of the study. The body weight variations between the mice on Day 1 did not exceed ±20% of the mean weight
Animal Source: Jackson Laboratories, Bar Harbor, ME 04609

Housing: Animals were group housed 5 per cage upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory
Animals". The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
Lighting: 12 hours light/12 hours dark
Room temperature: 26 to 29°C
Relative humidity: 16 to 76% in aeroneg enclosure but animals were housed in micro-isolator cages.
Food: Animals had access to Harlan Teklad Certified Rodent Chow 2016C ad libitum. No contaminants were known to be present in the certified diet at levels that
would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer.
Water: Tap water was available ad libitum, via water bottles. The water is routinely analyzed for contaminants. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study.
Acclimation: Study animals were acclimated to their housing for 14 days prior to their first day of dosing

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Group 1 - 0%
Group 2 - 25%
Group 3 - 50%
Group 4 - 100%

No. of animals per dose:

5

Details on study design:

DOSING
Route: Topically on the dorsal surface of both ear
Frequency: Once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent (± 3 hours) during the dosing phase.
Procedure: A volume of 25 uL/ear was applied using a micropipette.

IN-LIFE OBSERVATIONS
Mortality/Morbidity: Daily on Days 1 to 6
Clinical observations: Observations were performed prior to dose administration and immediately following dose administration on Days 1-3. Clinical observations
were also performed once daily on Days 4-6. Particular attention was given to the application sites. Any significant alterations to the application sites, and the general appearance of the pinnae, including build up of test article, was recorded.
Dermal irritation: Animals were examined daily for signs of erythema and edema. Irritation was scored and recorded using the Draize scoring system. Scoring was performed prior to dosing on Days 1-3.
Body weight: Animals were weighed on Days 1 and 6.

METHOD OF PERFORMANCE
Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and 3. Approximately 24±3 hours separated each application of test article. On Day 6, the mice were injected i.v. with 20 uCi of 3H-thymidine in 250 uL of sterile saline. Five hours later the mice were euthanized by C02 asphyxiation and the draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 4-5°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA
was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a beta-scintillation counter and
reported as DPM per mouse.

Positive control substance(s):

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

Statistics:

All data were collected manually except for the data generated by the beta-scintillation counter (Beckman LS 6000 SC). SYSTAT version 9.01, developed by SPSS, Inc was used for statistical analysis. The mean DPM and standard error (sem) for each group was determined. Increases in 3H-thymidine incorporation relative to the vehicle-treated control was derived for each group and recorded as stimulation indices (SI). The criterion for a positive response was that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.
Body weights and body weight changes on Days 1 and 6 were also evaluated using SYSTAT version 9.01, developed by SPSS, Inc. The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
Individual DPM values were analyzed by log transformation (base 1 0) of the data. The evaluation of the equality of means for the DPM was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.

Results and discussion

Positive control results:

SI (35%) = 7.1

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.3

Test group / Remarks:

25%

Remarks on result:

other: not sensitizing

Results 2

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

50%

Remarks on result:

other: not sensitizing

Results 3

Key result

Parameter:

SI

Value:

ca. 1.3

Test group / Remarks:

100%

Remarks on result:

other: not sensitizing

Cellular proliferation data / Observations:

MORTALITY AND CLINICAL OBSERVATIONS
There was no mortality and all animals appeared normal throughout the study.
DERMAL IRRITATION SCORES AND DOSE APPLICATION SITE OBSERVATIONS
No erythema or edema was noted in any of the mice in the vehicle group, those dosed with the test article at 25%, 50% or 100% (v/v) or the HCA group at 35%. The ears of all mice treated with the test article at 50% or 100% and those dosed with HCA appeared wet on Days 2-4. There were no other findings.
BODY WEIGHTS
Body weights at Day 1 and Day 6 and body weight changes from Day 1 to Day 6 were evaluated. Statistically significant differences in mean body
weights on Day 1 were observed for the 50%, 100% and HCA groups when compared to the mean body weight for the vehicle control for Day 1. As these
differences were only for the initial body weight they were not biologically relevant. There were no statistically significant differences observed between
any of the groups at Day 6. When the change in body weights were evaluated, the only statistically significant difference observed was in the positive control group. Therefore, the test article did not appear to cause any overt toxicity.
LOCAL LYMPH NODE ASSAY
At termination the lymph nodes in the mice treated with the vehicle and test article group at 25% (v/v) were normal in size and appearance. The lymph nodes from one of five treated with the test article at 50% and three of five treated with the test article at 100% were enlarged but otherwise normal in appearance. The lymph nodes from all five treated with HCA at 35% were enlarged but otherwise normal in appearance. The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.1. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p<0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

Any other information on results incl. tables

Group	Treatment	Dose	DPM (MEAN ±SEM)	SI (TEST/CONTROL RATIO)	Results*
1	Vehicle^a	-	908± 176	-	-
2	Test	25%	1198± 231	1.3	-
3	Test	50%	780 ±191	0.9	-
4	Test	100%	1208± 361	1.3	-
5	HCA	35%	6472± 684***	7.1	+

*- Test/control ratio of 3.0 or greater represents a positive result

a- AOO(4:1)W/V

***- Statistically significant difference when log DPM compared to the vehicle control groups (Group 1)(p<0.001)

Applicant's summary and conclusion

Interpretation of results:

other: not sensitizing

Conclusions:

A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index >= 3.0 is regarded as a positive response.
Therefore, based on the criteria of this study, treatment with test chemical at concentrations of 25%, 50% and 100% did not result in a stimulation index of 3.0 or greater and hence the substance at concentrations up to 100% did not have skin sensitizing activity.

Executive summary:

Mouse Local Lymphnode Assay was conducted to determine the allergenic potential of the test chemical. The assay was performed according to OECD 429 Guidelines. 25 Nulliparous and non-pregnant female CBA:J mice were used in the study.

A volume of 25 uL/ear of the test chemical at concentrations of 25%, 50% and 100% in acetone: olive oil(4:1) was applied topically on the dorsal surface of both ears once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent (± 3 hours) during the dosing phase.

On Day 6, the mice were injected i.v. with 20 uCi of 3H-thymidine in 250 uL of sterile saline. Five hours later the mice were euthanized by C02 asphyxiation and the draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 4-5°C. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a beta-scintillation counter and reported as DPM per mouse.

At termination the lymph nodes in the mice treated with the vehicle and test article group at 25% (v/v) were normal in size and appearance. The lymph nodes from one of five treated with the test article at 50% and three of five treated with the test article at 100% were enlarged but otherwise normal in appearance. The lymph nodes from all five treated with HCA at 35% were enlarged but otherwise normal in appearance. The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.1. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p<0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index >= 3.0 is regarded as a positive response.

Therefore, based on the criteria of this study, treatment with test chemical at concentrations of 25%, 50% and 100% did not result in a stimulation index of 3.0 or greater and hence the substance at concentrations up to 100% did not have skin sensitizing activity.