Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 08 March 2010 and 24 March 2010
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 relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
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
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15th September 2009 Date of signature: 26th November 2009
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :Lanolin Fatty Acids
Description : brown waxy solid
Batch number : 0000371886
Date received : 08 February 2010
Expiry date : 21 September 2010
Storage conditions :room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 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: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: ad libitum access to food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK)
- Water: ad libitum access to mains tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 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: Day 0 To: day of sacrifice - Day 6

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Preliminary screening test: 50% w/w in dimethyl formamide

Main test: 50, 25 and 10% w/w in dimethylformamide
No. of animals per dose:
Preliminary screening test: one mouse

Main test: four animals in total
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 dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

- Irritation: 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.

- Lymph node proliferation response: not recorded 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 of Test Material
For the purpose of the study, the test material was freshly prepared as a solution in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section.

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.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test material administration
Groups of four mice were treated with the test material at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. 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):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None provided

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 dimethyl formamide
Stimulation Index Result
15 5.16 Positive

Conclusion. α-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: see Remark
Remarks:
Concentration (% w/w) Stimulation in dimethylformamide index Vehicle n/a 10 1.42 25 1.77 50 2.35
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration (% w/w) Disintergrations in dimethylformamide per minute Vehicle 3985.00 10 5660.72 25 7038.21 50 9365.82

Any other information on results incl. tables

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 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 CommissionRegulation (EC) No. 440/2008

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of50% 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 asolutionindimethyl formamideat concentrations of50%,25% or10% w/w. A further group of four animals was treated withdimethyl formamidealone.

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
dimethyl formamide

Stimulation Index

Result

10

1.42

Negative

25

1.77

Negative

50

2.35

Negative

Conclusion. The test material was considered to be a non-sensitiserunder the conditions of the test.