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Description of key information

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear according to the OECD Guideline 429 (Skin sensitization: Local Lymph Node Asay)

The test item was considered to be not a sensitizer under the conditions of the test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 05 February 2008 and Experimental completion date: 26 February 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
- Test system: Mice, CBA/CaOlaHsd
- Rationale: Recognised as the recommended test system
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Number of animals for the pre-test: 2 females
- Number of animals for the main: study 16 females
- Number of animals per group: 4 females (nulliparous and non-pregnant)
- Number of test groups: 3
- Number of control (vehicle) groups: 1
- Age: 8 - 9 weeks (beginning of treatment)
- Identification: Single caging. The animals were distributed into the test groups at random and identified by cage number.
- Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry
- The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
- Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Environment: temperature 22 + 3°C
- relative humidity: 30-70%
- artificial light: 6.00 a.m. - 6.00 p.m.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50 microL (25 microL per ear) of the test item as a solution of acetone:olive oil (4+1) at a concentration of 10%, 25% and 50%.
No. of animals per dose:
4 females (nulliparous and non-pregnant) per group.
Details on study design:
Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 50% solution in acetone:olive oil (4+1).
To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 5, 10, 25, and 50 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. After the first application onwards the two highest doses (25 and 50%) induced reddening and loss of hair of the ear skin of all 4 animals of the group.
Nevertheless, excessive irritation was not observed.
The test item in the main study was assayed at 10, 25, and 50%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

Test Item Preparation
The test item was placed into a volumetric flask on a tared balance and acetone:olive oil (4+1) was quantitatively added.
The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Topical Application
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 10, 25, and 50% (w/v) in acetone:olive oil (4+1). The application volume, 25 Ql, was spread over the entire dorsal surface ( 8 mm) 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).

Administration of 3H-Methyl Thymidine
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 Ql of 79.4 QCi/ml 3HTdR (corresponds to 19.8 QCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, D-30827 Garbsen).
The draining lymph nodes were rapidly 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 Qm 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 approximately +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 of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, D-63110 Rodgau) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The - scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
Positive control results:
See "Any other information on results incl. tables" field.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Main test Estimation of the proliferative Response of Lymph Node Cells The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follow: Stimulation Indices of 1.83, 1.18 and 1.46 were determined with the test item at concentrations of 10, 25, and 50% 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.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: No data.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. After the first application onwards the highest dose (50%) induced loss of hair of the ear skin of all 4 animals of the group, nevertheless, reddening of the ear skin was not observed.

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.

DISCUSSION

To study a possible contact allergenic potential, three groups each of four female mice were treated daily with the test item at

concentrations of 10, 25, and 50% (w/v) in acetone:olive oil (4+1) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil (4+1)) only. Five days after the

first topical application the mice were injected intravenously into a tail vein with radiolabelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes 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 3H-methyl thymidine measured in a scintillation counter.

The animals did not show signs of systemic toxicity during the course of the study and no cases of mortality were observed. After the first application onwards the highest dose (50%) induced loss of hair of the ear skin of all 4 animals of the group but signs of irritation

did not occur.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.83, 1.18 and 1.46 were determined with the test item at concentrations of 10, 25, and 50% 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.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was not a skin sensitiser in this assay under the described conditions.
Executive summary:

In the study the test item 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 using test item concentrations of 10, 25, and 50%.

The animals did not show signs of systemic toxicity during the course of the study and no cases of mortality were observed. After the first application onwards the highest dose (50%) induced loss of hair of the ear skin of all 4 animals of the group but signs of irritation

did not occur.

In this study Stimulation Indices (S.I.) of 1.83, 1.18 and 1.46 were determined with the test item at concentrations of 10, 25, and 50% in acetone:olive oil (4+1), respectively.

The test item was not a skin sensitiser in this assay under the described conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A study was performed to assess the potential of the test item to induce contact allergenic according to OECD 429.

The test item was dissolved in acetone: olive oil (4 +1) at concentrations of 10, 25 and 50%.

The animals did not show signs of systemic toxicity during the course of the study and no cases of mortality were observed.

After the first application onwards the highest dose (50%) induced loss of hair of the ear skin of all 4 animals of the group but signs of irritation did not occur.

In this study Stimulation Indices (S.I.) of 1.83, 1.18 and 1.46 were determined with the test item at concentrations of 10, 25, and 50% in acetone:olive oil (4+1), respectively.

The test item was not a skin sensitiser in this assay under the described conditions.

Justification for classification or non-classification

Based on the above stated assessment of the skin sensitisation potential, the substance does not needs to be classified as Skin sensitiser according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.