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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:
2008-04-02 to 2008-04-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, adopted 2002-04-24
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)
Remarks:
signed 2007-10-15
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 17 to 21 g
- Housing: animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (ad libitum): Certified Ran and Mouse Diet
- Water (ad libitum): mains tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Relative humidity: 30 to 70 %
- Air exchanges: approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 %, 10 % or 5 % v/v in acetone/olive oil
No. of animals per dose:
4 female mice
Details on study design:
RANGE FINDING TESTS:
A preliminary screening test was performed using three mice, one mouse per test material concentration. The mice were treated by daily application of 25 µL of the undiluted test material or the test material at a concentration of 50 % or 25 % v/v in acetone/olive oil, to the dorsal surface of each ear for up to three consecutive days (Days 1, 2, 3). The mice were observed twice on Day 1 and pre-dose on Day 2 and the surviving mice were observed post dose on Day 2 and twice on Day 3. The remaining mouse was observed once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mouse on Day 6. The bodyweights of the mice that were humanely killed were recorded immediately prior to termination, except for that of one animal which was not recorded due to technician error.
Results:
The animal treated with the undiluted test material and the animal treated with the test material at a concentration of 50% v/v in acetone/olive oil were humanely killed, on Day 2 and Day 3 respectively, due to the occurrence of clinical signs of toxicity. Clinical signs of toxicity noted were hunched posture, lethargy, tiptoe or splayed gait, ptosis, and increased activity. Bodyweight loss and mild redness were also noted.

No signs of systemic toxicity were noted in the animal treated with the test material at a dose level of 25 % v/v in acetone/olive oil.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
The test material was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1, except for on one occasion during the preliminary screening test when due to technician error it was prepared in acetone/olive oil 2:1. This deviation from the protocol was considered not to affect the purpose or integrity of the study. This vehicle was chosen as it produced the most suitable formulation at the required concentration.
Groups of mice were treated with the test material at concentrations of 25 %, 10 % or 5 % v/v in acetone/olive oil. The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1,2, 3).

A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Five hours following the administration of 3HTdR all mice were killed and he draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

A single cell suspension of pooled lymph node cells was prepared, rinsed with PBS, and transferred to a centrifuge tube.The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % trichloroacetic acid (TCA).

After approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fliud (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then mesured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Please refer to "details on study design" above
Positive control results:
The stimulation index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
15 % v/v in acetone/olive oil 4:1:
- SI: 10.91
α-hexyl cinnamic aldehyde, Tech, 85 % was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A stimulation index of less than 3 was recorded for the three concentrations of the test material (25 %, 10 % and 5 % v/v in acetone/olive oil). Stimulation index: - 5 % v/v concentration: 1.19 - 10 % v/v concentration: 2.08 - 25 % v/v concentration: 2.97
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
- Vehicle: 9071.86 (dpm); 1133.98 (dpm/node) - 5 % v/v concentration: 10757.75 (dpm); 1344.72 (dpm/node) - 10 % v/v concentration: 18833.94 (dpm); 2354.24 (dpm/node) (results based on pooled treatment group approach) - 25 % v/v concentration: 26987.12 (dpm); 3373.39 (dpm/node)

Clinical observations and mortality data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test substance is not classified as skin sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation

One reliable animal study described in Bradshaw (2008) (OECD 429, GLP compliant) is considered to be reliable without restrictions. The substance was determined not to be a skin sensitiser.


Migrated from Short description of key information:
Skin sensitisation: not sensitising (OECD 429, GLP compliant)

Justification for selection of skin sensitisation endpoint:
GLP guideline study conducted with the test item

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

The reference Bradshaw (2008) is considered as the key study on skin sensitisation and will be used for classification. The overall sensitisation results are as follows:

Local lymph node assay in mice

SIs of less than 3.0 (1.19 - 2.97) were observed at all test concentrations of the test item. Thus, the classification criteria acc. to Regulation (EC) 1272/2008 as skin sensitiser are not met and the test item does not have to be labelled as such.