Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 05 January 2009 and 27 January 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
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:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK
- 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 (e.g. ad libitum): Free access to food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, 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 target ranges of 19 to 25°C
- Humidity (%): The relative humidity was controlled to remain within target ranges of 30 to 70%
- Air changes (per hr): 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: 0 To: 6

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary Screening Test - The mouse was treated with the test material at a concentration of 10% w/w in actone/olive oil 4:1

Main Test - Groups of four mice were treated with the test material at concentrations of 10%, 5% or 2.5% w/w in acetone/olive oil 4:1.
No. of animals per dose:
Preliminary Screening Test - 1
Main study - 4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the test material was freshly prepared as a solution/suspension in acetone/olive oil 4:1.
- Irritation: The mouse was treated by daily application of 25 µl of the test material at a concentration of 10% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was 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.
- Lymph node proliferation response: Not performed for preliminary Screening Test.


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:
Preparation:
For the purpose of the study, the test material was freshly prepared as a suspension in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Administration:
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 three consecutive days (Days 1, 2, 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.





Positive control substance(s):
other: alpha-Hexylcinnamaldehyde, Tech, 85%
Statistics:
None provided.

Results and discussion

Positive control results:
One group of five animals was treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further control group of five animals was treated with dimethyl formamide 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 dimethyl formamide: 15
Stimulation Index: 6.55
Result: 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: Concentration (% w/w) in acetone/olive oil 4:1 Vehicle: N/A 2.5%: 0.49 5%: 0.45 10%: 0.48
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Concentration (% w/w) in acetone/olive oil 4:1 Vehicle: 8794.60 dpm 2.5%: 4306.33 dpm 5%: 3957.94 dpm 10%: 4257.43 dpm

Any other information on results incl. tables

Preliminary Screening Test

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in acetone/olive oil 4:1

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in the table below.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (10%, 5% and 2.5% w/w in acetone/olive 4:1).

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
acetone/olive oil 4:1

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

8794.60

1099.33

na

na

2.5

4306.33

538.29

0.49

Negative

5

3957.94

494.74

0.45

Negative

10

4257.43

532.18

0.48

Negative


dpm=  Disintegrations per minute

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=      Stimulation Index of 3.0 or greater indicates a positive result

na =    Not applicable

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.

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:

Introduction. 

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 Directive 2004/73/EC

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% 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 four animals, were treated with 50 µl (25 µl per ear) of the test material as asuspensioninacetone/olive oil 4:1 at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated withacetone/olive oil 4:1alone.

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 are as follows:

Concentration (%w/w) in
acetone/olive oil 4:1

Stimulation Index

Result

2.5

0.49

Negative

5

0.45

Negative

10

0.48

Negative

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