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Description of key information

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:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with regulatory guidelines OECD429 and EU B.42 and to GLP
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:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Test animals: CBA/CaOlaHsd mice
- Source: Harlan NetherlandsB.V. Postbus 6174
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16-24g
- Housing: Single Makrolon type-2 cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3 C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light- 12 hours dark
Route:
epicutaneous, open
Vehicle:
other: acetone : olive oil (AOO) 4:1 (v/v)
Concentration / amount:
5, 10, 25 and 50%
Route:
other: none
Vehicle:
other: acetone : olive oil (AOO) 4:1 (v/v)
Concentration / amount:
5, 10, 25 and 50%
No. of animals per dose:
Four
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Once daily for three consecutive days
- Exposure period: 24 hours for each of the three days
- Test groups: 4
- Control group: 1
- Site: dorsal surface of both ear lobes
- Frequency of applications: once daily
- Duration: 3 days
- Concentrations: 5, 10, 25 and 50%
- Administraton of 3H-methyl thymidine: Five days after the first topical application, all mice were administered with 250 µl of 83.1 µCi/ml 3HTdR by intravenous injection via a tail vein.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25 and 50%
No. of animals per dose:
Four
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Not reported
- Irritation: Not reported
- Lymph node proliferation response: Not reported

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: Stimulation Index >3

TREATMENT PREPARATION AND ADMINISTRATION:
Topical (epidermal) application to dorsal surface of each ear lobe. A volume of 25 uL was spread over the entire dorsal surface (0-8mm) once daily for 3 consecutive days.

Five days after the first topical application, all mice were administered with 250uL of 83.1uCi/mL 3H methyl thymidine by intravenous injection into the tail vein. Five hours later animals were killed and draining lymph nodes excised and pooled for each experimental group i.e. 8 per group apart from group 3 (10%) where only 7 nodes were extracted.
Statistics:
Mean value and standard deviations for bodyweights only.
Parameter:
SI
Remarks on result:
other: Control - 1 5% -2.4 10% -2.7 25% - 1.6 50% - 2.2 Calculation of the EC3 value was not possible as an SI of 3 or higher was not reached in the study.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Control - 1381 (pooled); 173 per lymph node 5% - 3334 (pooled); 417 per lymph node 10% - 3304 (pooled); 471 per lymph node (only 7 lymph nodes collected) 25% - 2207 (pooled); 275 per lymph node 50% - 3099 (pooled); 387 per lymph node
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: EU CLP
Conclusions:
Not sensitising. No signs of local toxicity at the application site was noted.
Executive summary:

Four groups each of four female mice were treated daily with the test item at a concentration of 5, 10, 25 or 50% (w/v) in acetone : olive oil 4:1 (w/v) by topical application to the dorsal surface of each ear lobe for three consecutive days. A control group of four mice was treated with the vehicle. Five days after the first topical application, the mice were injected intravenously with radio-labelled thymidine. Approximately five hours after intravenous injection, the mice were sacrificed and 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 incorporation of 3H methyl thymidine measured in a beta scintillation counter.

Disintegrations per minute were determined per lymph node sample and Stimulation Indices (SI) relative to control group were calculated as follows:

5% SI= 2.4

10% SI = 2.7

25% SI = 1.6

50% SI= 2.2

Calculation of the EC3 value was not possible as a SI of 3 or higher was not reached in the study.

Decan-1,2-diol at concentrations up to 50% was found not to be a skin sensitiser.

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

In the key local lymph node assay (LLNA) conducted in accordance with OECD test guideline 429 and at dose concentrations up to 50%, a Stimulation Index of 3 was not achieved so the test substance was not classified as a skin sensitiser. In a second LLNA study, conducted partly in accordance with Test Guideline 429 but using a different evaluation method, based on lymph node cell counts rather than radioactivity counts as in the standard OECD test guideline, a positive result was recorded. This result was considered equivocal since the marginally positive result was recorded at the intermediate dose level only and this test was awarded a reliability rating of 2 because of the important deviations from the standard test protocol in terms of assessment of response.

The negative result in the key study was supported by a negative result in a clinical repeat insult skin patch study conducted to standard methodology and to GCP (dose concentration not reported) and also a negative result in a Guinea Pig Maximisation Test (GPMT) conducted using purified test material (using 1% intradermal induction dose and 5% topical induction and challenge dose concentrations). A second GPMT using technical grade material produced a positive sensitising response 48 hours post challenge for a 1% intradermal induction dose and a 25% challenge dose (25% was the highest non-irritant dose under the test conditions).


Migrated from Short description of key information:
1. Key LLNA (2004) - 5% SI = 2.4; 10% SI = 2.7; 25% SI = 1.6; 50% SI= 2.2. Calculation of the EC3 value was not possible as a SI of 3 or higher was not reached in the study.
Decan-1,2-diol at concentrations up to 50% was found not to be a skin sensitiser.
2. Supporting LLNA (2003) - 2% SI = 1.08; 10% SI = 1.59; 50% SI = 1.30. Cell count index threshold of 1.35 was only exceeded in the intermediate dose group.
Authors considered the conclusion to be that Decan-1,2-diol was "skin sensitising".
3. Supporting Guinea-pig maximisation test (2002) - using purified material but purity not reported. A 1% intradermal induction followed by 5% topical induction at 5 days and challenge at 21 days, decan-1,2-diol was considered non-sensitising as <30% of the animals were affected at 48 hours.
4. Supporting Guinea-pig maximisation test (2001) - using technical grade material, purity not reported. A 1% intradermal induction followed by a 25% topical induction at 5 days and challenge at 21 days, decan-1,2-diol was considered to be skin sensitising as >60% of the animals were affected at 48 hours.
5. Supporting repeat insult patch test in humans using only a small panel size of 55 subjects and a test substance concentration of 20%. Puriy of the test substance was not reported but considered technically pure material gave a non-sensitising result.

Justification for selection of skin sensitisation endpoint:
This study was selected as the key study for the following reasons:
1. It is a reliable (Klimisch 1) study
2. It is a GLP and Guideline (OECD429) study. Although no contemporary positive control group was incorporated into the study, the method was validated in the laboratory and strain of mouse using alpha hexyl cinnamic aldehyde in different vehicles and at concentrations of 3, 10 or 30% and demonstrating clear sensitising potential and it used the semi-quamtitative method of radio-active thymide incorporation.
3. The study was conducted using the highest concentration (50%) of a 99.5% pure test substance.
4. A clear negative result was obtained at all four concentrations tested with a Stimulation Index of <3 at 5, 10, 25 and 50 % concentrations.
The earlier LLNA (2003) was conducted using the less sensitive manual lymph node cell counting method and a less common strain of mouse. The assessment period was also after only four days in comparison with 8 days in the standard OECD429 test and the range of dose concentrations used was 2, 10 and 50%. The limit values for the Stimulation Index were also modified by the authors to a lower standard of 1.35. Despite these modifications an equivocal positive result was obtained at the intermediate dose concentration only with an SI of just 1.59. The 2001 Guinea-pig maximisation test was conducted with technical grade material and the 2002 study, using purified material, was conducted at a low challnege concentration (5%).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

On the basis of results obtained in the key LLNA using the highest concentrations of decan-1,2-diol, the test material may be considered as non-sensitising to skin.

No data was generated for assessment of respiratory sensitisation.