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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
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Identity: 2-Methylimidazol
- Appearance: Colorless solid
- Batch No.: 05649175L0
- BASF Substance No.: 02/0098-2
- Purity: 99.8 corr. area-% (determined by GC analysis)
- Stability in Solvent: Not indicated by the sponsor
- Storage: At room temperature, moisture protected
- Expiration Date: September 19, 2012

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., Postbus 6174, 5960 AD Horst, Netherlands
- Age at study initiation: pre-test 1 and 2: 10-11 weeks, main study: 9-10 weeks
- Weight at study initiation: 17.7-24.6 g
- Housing: Animals were group housed in Makrolon Type III cages, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany).
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:sterile water (7:3 v/v)
Concentration:
10, 25, and 50 % (w/w)
No. of animals per dose:
5
Details on study design:
Three groups each of five female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine; 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed. Both ears were punched at the apical area and the punches were immediately weighed pooled per animal. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. The suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Mean DPM per animal and S.I.
0 % (w/v): DPM = 225.4, S.I. = 1.00
5 % (w/v): DPM = 292.4, S.I. = 1.30
10 % (w/v): DPM = 635.8, S.I. = 2.82
25 % (w/v): DPM = 2035.6, S.I. = 9.03

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.14
Test group / Remarks:
50%
Parameter:
SI
Value:
1.07
Test group / Remarks:
25%
Parameter:
SI
Value:
1.02
Test group / Remarks:
10%
Parameter:
SI
Value:
1
Test group / Remarks:
0 %
Remarks on result:
other: negative control
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Concentration Disintegrations per minute
0% 202.9
10% 207.7
25% 217.9
50% 230.7

Any other information on results incl. tables

- Viability / Mortality: No deaths occurred during the study period.

- Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

- Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

- Lymph Node Weights and Cell Counts: The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weight or - cell count was not observed in any of the test item treated groups in comparison to the vehicle control group.

- Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was observed in the low and high dose group in comparison to the vehicle control group (p<0.05). As the observed increase was still within the range of historical vehicle control data for the ear weight, this was not regarded as biologically relevant.

Applicant's summary and conclusion