Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-04-30 to 2012-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline-conform study. For justification of read across see section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., 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 in Makrolon Type-II cages with standard softwood bedding.
- Diet: Teklad Global Rodent 2914C – Rezeptur 3255 GLP Batch 73/11 (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland), available ad libitum.
- Water: Community tap water from Itingen/Switzerland, available ad libitum.
- Acclimation period: 6 days prior to the start of the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 – 20.4°C
- 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)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by the guideline OECD 429.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 50% (w/v) in acetone:olive oil (4:1 v/v) and 100% on three consecutive days. In the pre-test clinical signs were recorded within approximately 1 hour and 24 ± 4 hours after each application as well as on the day corresponding to the day of preparation of the main experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: 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. (Stimulation Index) and 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:
The test item in the main study was assayed at 25 %, 50 % (w/v) in acetone:olive oil (4:1 v/v) and 100 %. The top dose is the highest technically achievable concentration while avoiding systemic toxicity and excessive local irritation. Test item was prepared freshly before dosing, no more than 4 hours prior to application to the ears. Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with the test item at concentrations of 25%, 50 % (w/v) in acetone:olive oil (4:1 v/v) or 100 %. The application volume of 25 μL was spread over the entire dorsal surface of each ear lobe (Ø about 8 mm) once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle.
Positive control substance(s):
other: none
Statistics:
For the body weight mean values and standard deviations were calculated.

Results and discussion

Positive control results:
not applicable

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The Stimulation Index was determined to be 1.22, 1.26 and 2.98 for the test item concentrations 25, 50 and 100 %, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: dpm/lymph node: group 1 (0 % test item): 140 group 2 (25 % test item w/v): 171 group 3 (50 % test item w/v): 176 group 4 (100 % test item w/v): 416

Any other information on results incl. tables

A dose-response relationship was not observed. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period. The body weights of the animals, recorded at the start of acclimatisation, prior to the first application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study S.I. of 1.22, 1.26 and 2.98 were determined with the test item dissolved in acetone:olive oil (4:1 v/v) at concentrations of 25%, 50% (w/v) and 100%, respectively. Therefore, the test substance was not concluded to be a 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. Stimulation indexes of 1.22, 1.26 and 2.98 were determined with the test item dissolved in acetone:olive oil (4:1 v/v) at concentrations of 25%, 50% (w/v) and 100%, respectively. As these are <3, the test substance was considered to be non sensitizing.