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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:
27 February 2006 and 30 March 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.
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/Ca CruBR)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood flakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70 %
Any occasional deviations from the above target ranges were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): approximately 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light


IN-LIFE DATES: From: 27 February 2006 To: 30 March 2006
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
Not applicable
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable
Positive control substance(s):
no
Remarks:
not applicable
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5 %, 10 % and 25 % w/w in acetone/olive oil 4:1
No. of animals per dose:
5 mice per dose
Details on study design:
RANGE FINDING TESTS:
One mouse was treated by daily application of 25 µl of the test material at a concentration of 25 % w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed twice daily on Day 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 the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
No signs of systemic toxicity were noted. Residual test material on the ears was noted one hour post dosing on Days 1, 2 and 3. Based on this information the dose levels selected for the main test were 5 %, 10 %, 25 % w/w in acetone/olive oil 4:1.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse. This 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 nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index (SI)).
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 5 mice were treated with the test material at concentrations of 5 %, 10 % or 25 % w/w 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 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 five mice received the vehicle alone in the same manner.

Five days following the first topical application of the test material (Day 6) all the 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, Amersham Biosciences UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for dpm and standard deviations where appropriate. Individual and group mean dpm 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 homogeneous datasets Dunnett’s Multiple Comparison test was used and for non-homogeneous datasets Dunnett’s T3 Multiple Comparison Method was used.
Positive control results:
Latest Positive Control Study for the Local Lymph Node Assay:

A study was performed to assess the sensitivity of the strain of mouse used in this study to a known sensitiser.

Test Material: α-Hexylcinnamaldehyde, Tech, 85 %
Study dates: 29 June to 5 July 2005

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 2.24 Negative
10 1.94 Negative
25 8.23 Positive

α-Hexylcinnamaldehyde, Tech 85 % was considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the three concentrations of the test material (5 %, 10 % and 25 % w/w in acetone/olive oil 4:1). See below for full results table.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See below for results table

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

 

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

 

Individual Dpm’s and Stimulation Index (SI)

 

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

Animal Number

Dpm / Animala

Mean Dpm / Animal (Standard Deviation)

Stimulation Index (SI)b

Result

Vehicle

1-1

1-2

1-3

1-4

1-5

2133.24

854.88

1938.82

2217.23

2247.17

1878.27 (±584.59)

N/A

N/A

5

2-1

2-2

2-3

2-4

2-5

2462.28

959.51

1506.11

2354.10

2446.57

1945.71 (±680.13)

1.04

Negative

10

3-1

3-2

3-3

3-4

3-5

2213.57

2489.95

1427.49

1819.28

1397.96

1869.65 (±480.52)

1.00

Negative

25

4-1

4-2

4-3

4-4

4-5

1015.55

1461.90

774.87

794.27

788.61

967.04* (±294.05)

0.51

Negative

 

a = Total number of lymph nodes per animal is 2

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

N/A = Not applicable

* = Significantly different from control group p<0.05

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 Local Lymph Node Assay sensitization study (0656/0283) with the test substance in acetone/olive oil 4:1, twenty 8-12 week old female CBA/Ca mice were tested using the method of EC Directive 92/69/EEC, Method B42. The positive control material was α-Hexylcinnamaldehyde. No signs of ill health or toxicity were observed.

In this study, the test material produced no evidence of skin sensitisation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The potential of the test material to cause sensitisation was investigated a local lymph node assay conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

In the mouse local lymph node assay (LLNA), female CBA/Ca (CBA/Ca CruBR) mice were exposed to the test material at concentrations of 5, 10 and 25 % w/w in acetone:olive oil 4:1 (5 mice per dose). The positive control material was α-Hexylcinnamaldehyde. 

No signs of ill health or toxicity were observed and a stimulation index of less than 3 was recorded for the three concentrations of the test material.

Under the conditions of this study, the test material produced no evidence of skin sensitisation.


Migrated from Short description of key information:
Not sensitising (LLNA); OECD 429 and EU Method B.42

Justification for selection of skin sensitisation endpoint:
Only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.