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Description of key information

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Skin sensitisation

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Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
20th April - 16th August 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Study conducted on middle weight fraction of shale oils.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (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:
other: CBA/CaHsdRcc (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, CH-4414, Füllinsdorf / Switzerland
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 - 24 g
- Housing: Individually housed in Makrolon type-2 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted diet available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen, available ad libitum.
- Acclimation period: 20th - 26th April 2005

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

IN-LIFE DATES: From: To: 20th April - 3rd May 2005
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 and 100 % in vehicle.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 10, 25, 50 and 100 % solutions in vehicle.
- Irritation: No irritation effects
- Lymph node proliferation response: NDA

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:

The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle acetone/olive oil (4/1, v/v) was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer.
Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears.
Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 10 %, 25 %, 50 % in acetone/olive oil (4/1, v/v) and 100 % (undiluted).
The test item in the main study was assayed at three consecutive concentrations. The top dose is the highest technically applicable concentration while avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
Concentrations were in terms of material as supplied.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean and standard deviations of body weights were calculated.
Positive control results:
Stimulation indices of 2.4, 3.6 and 11.2 were obtained at concentrations of 5, 10 and 25 % alpha-hexylcinnamaldehyde in the vehicle. The test subsatnce was therefore found to be a skin sensitizer.

The EC3 value is the concentration of test substance at which a score of 3 on the S.I. index is achieved. In the case of alpha-hexylcinnamaldehyde, this was achieved at 7.5 %.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 25 % test substance in vehicle gave a measurement of 68765 50 % test substance in vehicle gave a measurement of 57268 100 % test substance gave a measurement of 48048
Key result
Parameter:
SI
Value:
15.3
Variability:
25% test substance
Test group / Remarks:
Group 2: 4 animals
Key result
Parameter:
SI
Value:
12.7
Variability:
50% test substance
Test group / Remarks:
Group 3: 4 animals
Key result
Parameter:
SI
Value:
10.7
Variability:
100% test substance
Test group / Remarks:
Group 4: 4 animals

No deaths occurred during the study period.

No clinical signs were observed in any animals of the control group. On the second and the third application days, a slight ear erythema was observed at both dosing sites in all mice of Group 3 (50 %) and Group 2 (25 %), persisting for a total of four or three days. Since the second application day, a slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (100 %, undiluted), persisting for the remainder of the in-life phase of the study.

The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Stimulation indices of 15.3, 12.7 and 10.7 were determined with the test item at concentrations of 25 %, 50 % in acetone/olive oil (4/1, v/v) and 100 % (undiluted), respectively. Shale Oil was therefore found to be a skin sensitizer.
Executive summary:

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

In order to study a possible contact allergenic potential of Shale Oil, three groups each of four female mice were treated daily with the test item at concentrations of 25 %, 50 % in acetone/olive oil (4/1, v/v) and 100 % (undiluted) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. The study was conducted in accordance with OECD 249 and to GLP standard. A control group of four mice was treated with the vehicle acetone/olive oil (4/1, v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled 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 β-scintillation counter.

Stimulation indices of 15.3, 12.7 and 10.7 were determined with the test item at concentrations of 25 %, 50 % in acetone/olive oil (4/1, v/v) and 100 % (undiluted), respectively. Shale Oil was therefore found to be a skin sensitizer.

Based on the rationale for read-across, it is considered acceptable to use this study to address the same endpoint for the heavy fraction of shale oil.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

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

In order to study a possible contact allergenic potential of Shale Oil, three groups each of four female mice were treated daily with the test item at concentrations of 25 %, 50 % in acetone/olive oil (4/1, v/v) and 100 % (undiluted) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. The study was conducted in accordance with OECD 249 and to GLP standard. A control group of four mice was treated with the vehicle acetone/olive oil (4/1, v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled 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 β-scintillation counter.

Stimulation indices of 15.3, 12.7 and 10.7 were determined with the test item at concentrations of 25 %, 50 % in acetone/olive oil (4/1, v/v) and 100 % (undiluted), respectively. Shale Oil was therefore found to be a skin sensitiser.

Based on the rationale for read-across, it is considered acceptable to use this study to address the same endpoint for heavy fraction of shale oil.


Migrated from Short description of key information:
In an LLNA study in female mice, stimulation indices of 15.3, 12.7 and 10.7 were determined with the test item at concentrations of 25 %, 50 % in acetone/olive oil (4/1, v/v) and 100 % (undiluted), respectively. Shale Oil, Middle Fraction was therefore found to be a skin sensitiser.

Justification for classification or non-classification

The substance should be classified as a Category 1 skin sensitiser with associated hazard phrase H317 in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No.1272/2008.