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Administrative data

Description of key information

Local Lymph Node Assay: The test item was considered to be a non-sensitiser under the conditions of the test.

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:
14 March 2012 to 27 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
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)
Remarks:
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 8 to 12 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. The animals were provided with environmental enrichment which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100, 50 and 25 %
No. of animals per dose:
4/dose
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/ irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for 3 consecutive days (Day 1, 2 3). The mouse was observed twice daily on Days 1, 2 and 3 and once on Days 4,5 and 6. Local irritation was scored daily. Any clinical of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on day 6.

The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6, any changes in the ear thickness were noted. Mean ear thickness changes increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for 3 consecutive days (Day 1, 2 and 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 4 mice received 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 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 20. Ci/ mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Value:
1.31
Test group / Remarks:
25%
Remarks on result:
other: non-sensitising
Parameter:
SI
Value:
1.52
Test group / Remarks:
50%
Remarks on result:
other: non-sensitising
Parameter:
SI
Value:
1.74
Test group / Remarks:
100%
Remarks on result:
other: non-sensitising
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 8625.33; 25 %: 11305.86; 50 %: 13138.66 ;100 %: 15037.68

Preliminary screening test

No signs of systemic toxicity, visual skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted.

Based on this information the undiluted test item and the test item at concentrations of 50 % and 25 % v/v in acetone/ olive oil 4:1 were selected for the main test.

Main test

Estimation of the proliferative response of lymph node cells

Disintegrations per minute, disintegrations per minute/ node and Stimulation index

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

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

8625.33

1078.17

na

na

25

11305.86

1413.23

1.31

Negative

50

13138.66

1642.33

1.52

Negative

100

15037.68

1879.71

1.74

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

The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

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 of animals over the same period.

Current positive control study for the local lymph node assay

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

Stimulation Index

Result

25

5.76

Positive

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

Interpretation of results:
GHS criteria not met
Conclusions:
The sensitising potential of the test item was assessed according to OECD guideline 429. The test item was considered to be a non-sensitiser.
Executive summary:

Introduction.

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the OECD Guideline No. 429 "Skin Sensitisation: Local Lymph Node Assay".

Methods:

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.

Three groups of four CBA/Ca strain mice were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/ olive oil 4:1 at concentrations of 50 % or 25 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

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 (% v/v) in acetone/olive oil 4:1

Stimulation Index

Result

25

1.31

Negative

50

1.52

Negative

100

1.74

Negative

Conclusion:

The test item was considered to be a non-sensitiser under the conditions of the test.

 

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

A skin sensitisation study was conducted in the mouse in accordance with GLP and OECD Guideline 429. Three groups of 4 CBA/Ca strain mice were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/ olive oil 4:1 at concentrations of 50 % or 25 % v/v. 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 1.31 for the solution at concentration 25 % v/v, 1.52 for the solution at concentration 50 % v/v and 1.74 for the undiluted test item. Based on these results the test item was not considered to be a sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation: The available study has been assigned reliability 1 and is considered as acceptable for classification.Under the conditions of this study the substance was found to be a non-sensitiser. As such, the test item can be considered to be non-classified.