Benzyl methacrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-01-16 to 2007-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 24 April 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate dated 6 January 2005

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl methacrylate

EC Number:

219-674-4

EC Name:

Benzyl methacrylate

Cas Number:

2495-37-6

Molecular formula:

C11H12O2

IUPAC Name:

benzyl methacrylate

Test material form:

liquid

Specific details on test material used for the study:

Name of test material (as cited in study report): Benzyl methacrylate; 2-Propenoic acid, 2-methyl-, benzenemethyl ester

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- CBA/CaOlaHsd mice, nulliparous, non-pregnant
- Age at study initiation: 7-8 weeks (at beginning of acclimatisation)
- Weight at study initiation: 18.9 g ± 1.2 g
- Housing: single; Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 30-72%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2007-02-07 To: 2007-02-13

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

-non-GLP pretest (test substance): 12.5%; 25%; 50%; 100%
-Main study (test substance): 25%; 50%; 100%
-Positive control: α-Hexylcinnamaldehyde in acetone: olive oil (4+1)
-Negative control: vehicle

No. of animals per dose:

4 female mice

Details on study design:

RANGE FINDING TESTS:
Concentrations were tested on two mice on one ear each.
- Compound solubility:
in water: virtually insoluble at 20°C
in vehicle: miscible with most organic solvents
- Irritation: no irritation effects were observed at any concentration after single application (even not at 100%).
- Lymph node proliferation response: no data

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
Each group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 25, 50 and 100% (w/v) in acetone: olive oil (4+1). The application volume (25 µL) was spread over the entire dorsal surace (diam. approx. 8 mm) of each ear lobe once daily for three consecutive days. The control group of mice was treated with an equivalent volume of the vehicle alone.
5 days after the 1st topical application, all mice were administered with 250 µL of 80.5 µCi/mL 3HTdR (=20.13 µCi 3HTdR per mouse) by intravenous injection via a tail vein. 5 hours after this injection, all mice were euthanised.
The draining lymph nodes were excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5% trichloroacetic acid (approx. 3 mL) and incubated at approx. +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL scintilation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
The proliferate response of lymph node cells is expressed as DPM/node and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation background DPM was subtracted from test and control raw data. Background DPM was measured with a duplicate set of 1 mL 5% trichloroacetic acid.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE::
Two criteria needed to be fulfilled:
(1) exposure to at least one concentration of the test item resulted in incorporation of 3H-methyl thymidine (3HTdR) being at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (based on investigations performed with a wide range of chemicals, the indication of sensitizing activity was set to S.I.=3)
(2) data are compatible with conventional dose response (although allowance must be made (especially. at high topical concentrations) for either local toxicity or immunological suppression)

Positive control substance(s):

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

Statistics:

none

Results and discussion

Positive control results:

A positive control performed with alpha-Hexylcinnamaldehyd in December 2006 resulted in an S.I. of 2.04 at 5% (w/v) alpha-Hexylcinnamaldehyd, 6.31 at 10% and 12.45 at 25%. An EC3 of 6.1% (w/v) was calculated.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

4.28

Test group / Remarks:

25 %

Remarks on result:

other: EC3 could not be calculated due to all S.I. being above 3.

Results 2

Parameter:

SI

Value:

6.92

Test group / Remarks:

50 %

Remarks on result:

other: EC3 could not be calculated due to all S.I. being above 3.

Results 3

Parameter:

SI

Value:

6.57

Test group / Remarks:

100 %

Remarks on result:

other: EC3 could not be calculated due to all S.I. being above 3.

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle control group (negative control with vehicle, only): 794.2 DPM per lymph node (8 lymph nodes of pooled animals of a dose group assessed) Test substance: 25% DPM/lymph node: 3403 50% DPM/lymph node: 5498.5 100% DPM/lymph node: 5219.6

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In this local lymphnode assay Benzyl methacrylate is a dermal sensitiser and attained the criteria of Skin sens Category 1 B.

Executive summary:

In a dermal sensitisation study according to OECD Guideline 429 (adopted 24 April 2002) with BNMA in acetone:olive oil (4+1, v/v), groups of 4 female CBA/CaOlaHsd mice were tested using the LLNA method.

STIMULATION INDICES (S.I.) of 4.28, 6.92 and 6.57 were determined with the test substance at concentrations of 25%, 50% and 100% (w/v) in acetone:olive oil (4+1, v/v), respectively. The positive control substance was α-Hexylcinnamaldehyde, which gave an EC3 at 6.1% (w/v).

A result is regarded as positive when the S.I. is ≥3.

Based on these criteria, the test substance was found to be a sensitiser at any concentration tested. However, the EC3 could not be calculated due to all calculated S.I.s being above 3. Accordingly, the EC3 is estimated to be below a concentration of the test substance of 25% (w/v).

In this local lymphnode assay Benzyl methacrylate is a dermal sensitiser and attained the criteria of Skin sens Category 1 B.

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