Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008/11/03-2008/11/18
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.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
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:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Harlan Laboratories UK Limited, Bicester, Oxon, UK.
- Age at study initiation:
8-12 weeks
- Weight at study initiation:
15-23 g
- Housing:
Suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum):
Ad libitum
- Water (e.g. ad libitum):
Ad libitum
- Acclimation period:
at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19-25 deg C
- Humidity (%):
30-70%
- Air changes (per hr):
Ca. 15
- Photoperiod (hrs dark / hrs light):
12 hours dark/12 hours light

IN-LIFE DATES: Not stated.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Preliminary screening test: 25%
Main test: 5%, 10% and 25%
No. of animals per dose:
Preliminary test:
25 micro.l at 25% concentration - 1 female animal
Main test:
25 micro.l at 5% concentration - 4 animals
25 micro.l at 10% concentration - 4 animals
25 micro.l at 25% concentration - 4 animals
Vehicle control - 4 animals
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 sulphoxide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
- Irritation:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse.
- Lymph node proliferation response:
Please see table 2 below.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
Not stated.
- 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:
Preliminary test:
The mouse was treated by daily application of 25 µl of the test material at a concentration of 25% w/w in dimethyl sulphoxide, 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. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
Main test:
Groups of four mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in dimethyl sulphoxide. 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.


RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
- Lymph node proliferation response:


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
LLNA
- 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:
-Preliminary test:
The mouse was treated by daily application of 25 µl of the test material at a concentration of 25% w/w in dimethyl sulphoxide, 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. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
-Main test:
Groups of four mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in dimethyl sulphoxide. 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:
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".

Results and discussion

Positive control 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
dimethyl sulphoxide Stimulation Index Result
5 1.88 Negative
10 3.67 Positive
25 4.93 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: See remarks section
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See remarks section

Any other information on results incl. tables

Table2              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
dimethyl sulphoxide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

6527.46

815.93

na

na

5

7761.64

970.21

1.19

Negative

10

6301.11

787.64

0.97

Negative

25

22136.11

2767.01

3.39

Positive

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

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a 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)

§        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 of25% 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 sulphoxideat concentrations of25%,10% or5% w/w. A further group of four animals was treated withdimethyl sulphoxidealone.

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 sulphoxide

Stimulation Index

Result

5

1.19

Negative

10

0.97

Negative

25

3.39

Positive

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