Methyl benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2012-04-11 to 2012-06-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl benzoate

EC Number:

202-259-7

EC Name:

Methyl benzoate

Cas Number:

93-58-3

Molecular formula:

C8H8O2

IUPAC Name:

methyl benzoate

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories BV, Maasheseweg 87c, 5804 AB Venray / Netherlands
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 16.2-23.9 g
- Housing: group housing
- Diet (e.g. ad libitum): Teklad Global Rodent 2914C – Rezeptur 3255 GLP Batch 73/11 (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland), available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen/Switzerland, available ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-20.4
- Humidity (%): 37.4-62.2
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50, 100 % (w/v) in vehicle

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration which can be technically used was a 75% (w/w) solution in acetone:olive oil (4:1 v/v). As the test item is a well pipettable liquid, it was also tested neat (100%).
- Irritation: At the tested 50 and 100 % concentrations, the animals did not show any signs of irritation.
- Lymph node proliferation response: is expressed as the number of radioactive disintegrations per minute per lymph node (dpm/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of the test groups relative to that recorded for the control group (Stimulation Index S.I.). Before dpm/lymph node values were determined, the mean scintillation-background dpm was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Topical application / Administration of 3HTdR / Determination of incorporated 3HTdR
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the S.I.
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: Test item preparation was made freshly before each dosing occasion, no more than 4 hours prior to application to the ears.

Positive control substance(s):

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

Statistics:

For the body weight mean values and standard deviations were calculated.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.22

Test group / Remarks:

25% test item

Results 2

Key result

Parameter:

SI

Value:

1.26

Test group / Remarks:

50% test item

Results 3

Key result

Parameter:

SI

Value:

2.98

Test group / Remarks:

100% test item

Any other information on results incl. tables

The following results were obtained for methyl benzoate using acetone:olive oil (4:1 v/v) as vehicle:

Test item concentration % (w/v)	Groups	Measurement dpm	Calculation			Result SI
			dpm-BG	Number of lymph nodes isolated	dpm / lymph node^a)
--	BG I	1	--	--	--	--
--	BG II	1	--	--	--	--
--	Group 1	1120	1117	8	140	--
25	Group 2	1368	1365	8	171	1.22
50	Group 3	1408	1405	8	176	1.26
100	Group4	3330	3327	8	416	2.98

BG = Background (1 mL 5 % trichloroacetic acid) in duplicates

SI = Stimulation Index (Ratio = dpm/lymph node test item treated group / dpm/lymph node Group 1)

a) = Since the lymph nodes of the animals of a dose group were pooled, dmp/lymph node was determined by dividing the measured value by the number of lymph nodes pooled.

The EC3 value was not determined as no SI value was equal or above 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item methyl benzoate was not a skin sensitizer in this assay.

Executive summary:

In order to study the possible contact allergenic potential of Methyl Benzoate, three groups of four female mice each were treated daily with the test item at concentrations of 25%, 50% (w/w) in acetone:olive oil (4:1 v/v) and 100% by topical application to the dorsum of each ear lobe for three consecutive days. A control group of four female mice was treated with the vehicle acetone:olive oil (4:1 v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter. All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period. The results obtained [Stimulation Index (S.I.)] are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

	Test item concentration % (w/v)	SI
Group 2	25	1.22
Group 3	50	1.26
Group 4	100	2.98
EC3: -- %

A dose-response relationship was not observed. A calculation of the EC3 value was not performed. In conclusion, the EC3 value was not determined as no SI value was equal or above 3.