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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2010 to 13 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

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
Principles of method if other than guideline:
A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of OECD 429.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Description: extremely pale yellow liquid
- Storage conditions: approximately 4 °C in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Female CBA/Ca (CBA/CaOlaHsd) strain mice were used. The animals were nulliparous and non pregnant.
- Age at study initiation: eight to twelve weeks old.
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food was allowed throughout the study.
- Water: Free access to mains tap water was allowed throughout the study.
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 to 70 %
- Air changes: rate of air exchange was approximately fifteen changes per hour
- Photoperiod: lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Groups of four mice were treated with the test material at concentrations of 0.5, 0.25 or 0.10 % v/v in acetone/olive oil 4:1.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: For the purpose of the study, the test material was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test material was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- Irritation: no data
- Lymph node proliferation response:
The animals treated with the test material at concentrations of 50, 10 or 1 % v/v in acetone/olive oil 4:1 were humanely killed on Day 2 or Day 3, due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted were hunched posture, lethargy, ataxia, decreased or increased respiratory rate, laboured respiration, ptosis, pilo-erection, hypothermia and splayed gait. Bodyweight loss was noted in the animals treated with the test material at concentrations of 50 or 1 % v/v in acetone/olive oil 4:1.
No signs of systemic toxicity were noted in the animal treated with the test material at a concentration of 0.5 % v/v in acetone/olive oil 4:1.
Based on this information the dose levels selected for the main test were 0.5, 0.25 and 0.10 % v/v in acetone/olive oil 4:1.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-Methyl Thymidine Administration
- 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:
Groups of four mice were treated with the test material at concentrations of 0.5, 0.25 or 0.10 % v/v in acetone/olive oil 4:1. 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 µl of the appropriate concentration of the test material to the dorsal surface of each ear for two consecutive days (Days 1, 2). The surviving mice were similarly treated on the following day (Day 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.
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 or vehicle (Day 6) all surviving 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, ARC UK Ltd) giving a total of 20 µCi to each mouse.


OBSERVATIONS:
Clinical observations: All animals were observed twice daily on Day 1 and 2 and pre-dose on Day 3. The surviving mice were observed post dose on Day 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). The bodyweight of the mouse that was humanely killed was recorded immediately prior to termination.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no data

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 is as follows:

Concentration (% v/v) in acetone/olive oil 4:1: 15
Stimulation Index: 3.12
Result: Positive

α Hexylcinnamaldehyde, tech., 85 % was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
5.8
Test group / Remarks:
0.10 % (v/v)
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
13.39
Test group / Remarks:
0.25 % (v/v)
Remarks on result:
other: Positive
Key result
Parameter:
SI
Value:
14.87
Test group / Remarks:
0.5 % (v/v)
Remarks on result:
other: Positive
Cellular proliferation data / Observations:
One animal treated with the test material at a concentration of 0.5 % v/v in acetone/olive oil 4:1 was humanely killed, pre dose on Day 3, due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted in this animal were hunched posture, lethargy and ptosis. Mild redness to the ears was also noted in this animal.

No signs of systemic toxicity were noted in the surviving test or control animals during the test. Mild redness to the ears and neck was noted on Days 4 and 5 in surviving animals treated with the test material at a concentration of 0.5 % v/v in acetone/olive oil 4:1.

Bodyweight changes of the surviving 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:
other: EU Criteria: H317: May cause an allergic skin reaction
Conclusions:
The test material was considered to be a sensitiser under the conditions of the test.
Executive summary:

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

 OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

 Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 0.5% v/v in acetone/olive oil 4:1, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 0.5%, 0.25% or 0.10% v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index 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/olive oil 4:1 Stimulation Index Result
0.1 5.8 Positive
0.25 13.39 Positive
0.5 14.87 Positive

The test material was considered to be a sensitiser under the conditions of the test.