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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) and in accordance with GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Guideline followed.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report)
- Physical state: White powder
- Analytical purity: 99.5%
- Storage condition of test material: Room temperature in the dark.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Age at study initiation: 8-12 weeks (All the females were nulliparous and non-pregnant)
- Weight at study initiation: 15-23 g.
- Fasting period before study: overnight fasting immediately before dosing.
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 Deg C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light.

Study design: in vivo (non-LLNA)

Induction
Concentration / amount:
Not applicable
Challenge
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable

Study design: in vivo (LLNA)

Vehicle:
other: 1% pluronic L92 distilled water
Concentration:
Concentration (%w/w) in 1% pluronic L92 distilled water:
5%, 10%, 25%
No. of animals per dose:
5 mice
Details on study design:
Groups of five mice were treated with the test item at concentrations of 25%, 10% and 5% w/w in 1% pluronic L92 in distilled water. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 micro L of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

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

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 micro L of phosphate buffered saline (PBS) containing 3H-methyl thymidine giving a total of 20microCi to each mouse.

Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 ml of PBS was added tothe lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The 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).


Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by p-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. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Clinical Observations: 
All animals were observed twice daily on Days1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of illhealth during the test were recorded.  Due to a technician error the Day1 pre-dose observation was performed 1 hour post dose. This deviation from the Study Plan was considered not to affect the purpose or integrity of the study,
  
Bodyweights: 
The body weight of each mouse was recorded on Day1(prior to dosing) and Day 6 (prior to termination).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Results and discussion

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 was found to be 6.77
a-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser (positive) under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 0.92, 0.95 and 0.60 for 5%, 10% and 25% respectively - NEGATIVE.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/Animal: 1287.34, 1182.16, 1218.29, 776.86 for vehicle, 5%, 10% and 25% respectively - NEGATIVE.

Any other information on results incl. tables

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio o f3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitiser if at least one concentration of the test item results in a three fold or greater increase in 3HTdR incorporation compared to control values.  Any test item failing to produce a three fold or greater increase in 3HTdR incorporation will be classified as a"non-sensitiser".

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

A study was performed to assess the skin sensitization potential of the test item inthe CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method followed was similar to OECD TG No.429.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentrations of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the local lymph node assay. Three groups, each of 5 animals, were tested with 50 microliter (25 microliter per ear) of the test item as a suspension in 1% pluronic L92 in distilled water at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with 1% pluronic L92 in distilled water.

Results: The stimulation index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 0.92, 0.95 and 0.60 for 5%, 10% and 25% respectively - NEGATIVE.

CONCLUSION: The test item was considered to be Non-sensitiser.