Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26th June 2006 to 20th July 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaBkl and CBA/Ca CruBR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
CBA/CaBkl strain mice were supplied by B&K Universal Ltd, Hull, UK
CBA/Ca CruBR strain mice were supplied by Charles River UK Limited, Margate, Kent, UK

- Age at study initiation:
At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 15 to 23g.

- Housing:
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.

- Diet (e.g. ad libitum):
Food (Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK) was allowed throughout the study.

- Water (e.g. ad libitum):
Free access to mains tap water was allowed throughout the study.

- Acclimation period:
At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
The temperature was controlled to remain within the target ranges of 19 to 25 deg C.

- Humidity (%):
The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr):
The rate of air exchange was approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light):
The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: From: 26th June 2006 To: 20th July 2006

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Each group was exposed to concentrations of 100% (undiluted), 50% or 25% v/v (in acetone/olive oil 4:1)
No. of animals per dose:
Groups of four mice were treated.
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using two mice. The mice were treated by daily application of 25 micro l of the undiluted test material and the test material at concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2 3). The mice were observed twice daily on Days 1, 2 and 3 and 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 on Day 6.

- Lymph node proliferation response:
Clinical observations, bodyweight and mortality data are give in Attachment 1.

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 25% and 50% v/v in acetone/olive oil 4:1 and 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

- 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(dpm/node) and as the ratio of 3HTdR incorporation in 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-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was used undiluted and also freshly prepared in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above. The vehicle determination record is included as Attachment 2.

Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guidelines.

The preliminary screening test suggested that the test material 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 material to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 micro l of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 microCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 microCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Clinical Observations: All animals were observed twice daily on Days 1, 2, and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Results and discussion

Positive control results:
The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration % v/v in acetone Stimulation Index (SI) Result
5 3.53 Positive
10 5.39 Positive
25 8.23 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the three concentrations of the test material (25% and 50% v/v in acetone/olive oil 4:1 and 100%)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Attachment 4 Table 2.

Any other information on results incl. tables

Clinical Observations and Mortality Data:

Individual clinical observations and mortality data for test and control animals are given in Attachment 4 Table 3.

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

Bodyweight:

Individual bodyweights and bodyweight changes for test and control animals are given in Attachment 4 Table 3.

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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

In a dermal sensitization study (LLNA) with U-donor 25% and 50%] in acetone/olive oil 4:1, four female, CBA/CaBkl and CBA/Ca CruBR strain mouse were tested using the standard LLNA method.

Alpha-Hexylcinnamaldehyde, Tech 85% was used as a positive control material.  

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

In this study, U-donor is not a dermal sensitizer