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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 14 September 2007 to 01 November 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
The relative humidity in the animal room was between approximately 30 - 85 % (instead of a maximum of 70%). This deviation had no detrimental impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
other: liquid

In vivo test system

Test animals

other: CBA/CaOlaHsd
Details on test animals and environmental conditions:
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst, The Netherlands
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 19.4 to 23.4 g
- Housing: Individually in Makrolon type-1 cages with wire mesh top (EHRET GmbH, D-79302 Emmendingen) and with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
- Diet: pelleted standard diet (Harlan Winlkelmann Gmbh, D-33178 Borchen), ad libitum
- Water: tap water (Gemeindewerke, D64380 Rossdorf), ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-85%
- Air changes (per hr): information not available
- Photoperiod (hrs dark / hrs light):6/6

IN-LIFE DATES: From 18 September 2007 to 02 October 2007

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
5, 10, 25, 50 and 100% (w/v) in acetone:olive oil (4+1)
No. of animals per dose:
4 per dose
Details on study design:
- Compound solubility and irritation:
A pre-test was performed on 2 mice to determine the highest non-irritant test concentration, or the highest technically applicable concentration. The data showed that the highest test item concentration, which could be technically used was 100%. At concentrations of 10, 25, 50 and 100%, the notified substance did not show any signs of irritation. Therefore, in the main assay, concentrations applied were 5, 10, 25, 50 and 100%.

- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
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 stimulation index and data are compatible with a conventional dose response (allowing for either local toxicity or immunological suppression).

Each test 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 5, 10, 25, 50, and 100% (w/v) in acetone:olive oil (4+1). The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µl of 78.2 µCi/ml 3H-methyl thymidine (corresponds to 19.6 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Prior to the first application of the test item and prior to treatment with 3HTdR.the ear thickness was determined using a micrometer .
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled per group (8 nodes/group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze. After washing two times with phosphate buffered saline, the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and transferred to plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
An aliquot of 50 µL of the cell suspension was diluted with an equal amount of trypan bule solution and the number of viable cells was determined using a Neubauer chamber. Results were expressed as cells per lymph node.
After the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed per animal using an analytical balance.
The proliferative 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The mean values and standard deviations were calculated in the body weight tables.

For the ear weights the ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Experiment performed in May 2007 usin gCBA/CaOlaHsd mice gave the following results for alpha-Hexylcinnamaldehyde in acetone:olive oil (4+1):
2.43 at 5%
4.07 at 10%
4.88 at 25%

In vivo (LLNA)

Resultsopen allclose all
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Any other information on results incl. tables

When cell counts were analysed for the same lymph node preparations, Stimulation indices (S.I.) of 1.08, 0.94, 1.14, 0.99 and 0.94 were determined at concentrations of 5, 10, 25, 50 and 100%, respectively.

The EC3 (concentration of the test material required to produce a 3-fold increase in draining lymph node cell proliferative activity) value could not be calculated since none of the tested concentrations induced a S.I. greater than 3.

Other observations:
The animals did not show any clinical signs during the course of the study and no case of mortality were observed. There were no signs of local irritation following application for 3 consecutive days.

No relevant increase in ear thickness and no statistically significant increase in ear weights were determined.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
In this study the stimulation indices (S.I.) of 1.12, 0.98, 0.76, 0.61 and 0.55 were determined with the test item at concentrations of 5, 10, 25, 50 and 100% in acetone:olive oil (4+1). The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.The test material Dimethyl 2-methylglutarate was found not to be a skin sensitiser under the described conditions.

Dimethy 2-methylglutarate is not classified as a skin sensitiser according to the criteria of Annex VI Directive 67/548/EEC and EU GHS
Executive summary:

In the study the test material Dimethyl 2-methylglutarate dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed in CBA/CaOlaHsd mice using test material concentrations of 5, 10, 25, 50, and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. No relevant increase in ear thickness and no statistically significant increase in ear weights were determined.

In this study Stimulation Indices (S.I.) of 1.12, 0.98, 0.76, 0.61, and 0.55 were determined for incorporation of 3H-thymidine with the test material at concentrations of 5, 10, 25, 50, and 100% in acetone:olive oil (4+1), respectively. When cell counts were analysed for the

same lymph node preparations Stimulation Indices (S.I.) of 1.08, 0.94, 1.14, 0.99, and 0.94 were determined.

Dimethyl 2-methylglutarate is not considered as a skin sensitiser according to the criteria of Annexe VI Directive 67/548/EEC and EU GHS.