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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 29 March to 12 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other 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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
N° 2011/40
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
EC Number:
934-058-1
IUPAC Name:
Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Reaction mass of N-[2-(2-Oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
- Storage condition of test material: at room temperature and protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: preliminary test: approx. 12 weeks old, Main study: 8 weeks old
- Weight at study initiation: 19.5 g (range 18.5 to 21.5g)
- Housing: by group of two (preliminary test) or four (main test) in polycarbonate cages (Tecniplast 1145T, 435 cm2) containing autoclaved sawdust (SICSA, Alfortville, France)
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 5776558, (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From 25 to 30 April 2012

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
batch No. BCBF9893V (Sigma)
Concentration:
Preliminary study: 10; 25; 50 and 100 %
Main study: 0; 5; 10; 25; 50 and 100 %
No. of animals per dose:
Preliminary study: 2
Main study: 4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:The first choice vehicle was a mixture of Acetone/Olive oil 4/1; v/v)(AOO). As unsatisfactory solubility of the test item was obtained in AOO (i.e. heterogeneous emulsion or suspension to the naked eye at the concentration of 50% was obtained), dimethylformamide (DMF) was used. A solution was obtained at the concentration of 50% in DMF.
- Irritation: Measurement of the ear thickness and observation of local reactions. No local reactions were observed in any animals up to the highest concentration (100%). The percentage of ear thickness increase compared to day 1 was less than 10% in all animals and at all concentrations. Therefore the test item is considered as non-irritant.
- Lymph node proliferation response: not examined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: the animals were allocated to the groups using a manual randomization procedure. A larger number of animals than necessary was acclimated to permit the selection and/or replacement of individuals. The animals were individually identified on the tail using an indelible marker (unique CIT identity number).
- Criteria used to consider a positive response: the test item is considered as a skin sensitizer when the SI for a dose group is >= 3 together with consideration of a dose-response relationship. Other relevant criteria such as radioactivity levels and ear thickness are also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION: On days 1, 2 and 3, a dose-volume of 25 µL of the control or dose formulation preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.
The maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an > 25% increase of the ear thickness.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
not required

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 3.53). The experiment was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.41
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.74
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
0.63
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.38
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.79
Test group / Remarks:
100

Any other information on results incl. tables

No unscheduled death occured during the study, and no clinical signs were observed in any animals. The body weight change of test item-treated animals was similar to that of control animals.

Table 7.4.1/1: Results of the proliferation assay (mean values)

Groups

Treatment and concentrations

Number of node per group

dpm per group

dpm per node

Stimulation index (SI)

Increase on ear thickness (% between day 1 and 6)

Irritation level

EC3value

3

Vehicle

8

5103

637.88

-

-4.81

-

-

4

Test item 5%

8

2117

264.63

0.41

-4.72

I

N/A

5

Test item 10%

8

3760

470.00

0.74

-5.71

I

6

Test item 25%

8

3229

403.63

0.63

-3.77

I

7

Test item 50%

8

7067

883.38

1.38

-3.88

I

8

Test item 100%

8

14214

1776.75

2.79

-7.41

I

9

HCA 25%

8

18008

2251.00

3.53

-

-

-

N/A: Not Applicable

dpm: Disintegrations per minute

HCA: α-hexylcinnamaldehyde

I: non-irritant (increase in ear thickness < 10%)

EC3 value: theoretical concentration resulting in a SI value of 3

Stimulation Index = dpm of treated group / dpm of control group

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide is not classified as a skin sensitiser in a Local lymph node assay in mouse according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and the Directive 67/548/EEC.
Executive summary:

In a local lymph node assay performed according to the OECD guideline No. 429 and in compliance with the GLP, 8-weeks old female CBA/J mice, were exposed to Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide (purity of 99.3%) undiluted or diluted in Dimethylformamide (DMF).

A preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two mice received the test item by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. No local reactions were observed in any animals up to the highest concentration (100%). The percentage of ear thickness increase compared to day 1 was less than 10% in all animals and at all concentrations. Therefore the test item is considered as non-irritant and the maximum concentration of 100% was chosen for the main study.

 

In the main study, 4 mice/concentration were topically treated with the test item at concentrations in DMF of 0; 5; 10; 25; 50 and 100 %. The topical application was performed on to the dorsal surface of both ears on days 1, 2 and 3 under a dose-volume of 25 µL. Additionally, one positive control group of four females received the positive control, α‑hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions. From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6.

After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR.

The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

No unscheduled death occurred during the study and no clinical signs were observed in any animals. The body weight change of test item-treated animals was similar to that of control animals. The threshold positive value of 3 for the SI was reached in the positive control group (SI = 3.53). The experiment was therefore considered valid.The stimulation index of the treated animals increased with the test item concentration, but was never higher or equal to 3 (max 2.79 for the 100% concentration). Dryness of the skin was observed on day6 in1/4 female treated at the concentration of 100%. No notable increase in ear thickness was observed at any tested concentrations.

 

Therefore under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide is not classified as a skin sensitiser in a Local lymph node assay in mouse according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and the Directive 67/548/EEC.

This study is considered as acceptable and satisfies the requirement for skin sensitisation endpoint.