Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern GLP study conducted in accordance with modern guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Purity of vehicle not specified in study report
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Purity of vehicle not specified in study report
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid
- Lot/batch No.: E0011-039E
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8–12 weeks old
- Weight at study initiation: 15–23 g
- Housing: Individuall housed is suspended solidfloor propylene 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


ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled to remain within target range of 19–25 deg C
- Humidity (%): Controlled to remain within target range of 30–70 deg C
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark (06:00–18:00)

Study design: in vivo (LLNA)

Vehicle:
other: butanone
Concentration:
0%, 10%, 25% and 50% w/w test material in butanone
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of the test material at 50% w/w in butanone on Days 1, 2, and 3, and further observed until Day 6. Any signs of toxicity, excessive local irritation or effects on bodyweight were noted.


MAIN STUDY
- 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). 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:
The mice were treated by daily application of 25 microlitres 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute 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 homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:
Two groups, each of five animals, were treated with 50 microlitres (25 microlitres per ear) of a-Hexylcinnamaldehyde, Tech, 85% as a solution in butanone at concentrations of 10% and 25% v/v. A further control group of five animals was treated with butanone alone.

The low dose and high dose produced Stimulation Indices of 2.86 (negative) and 5.74 (positive) respectively.

a-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:
The Stimulation Index for each group was: Concentration Stimulation Index 0 (vehicle control) N/A 10 2.12 25 2.36 50 2.75
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Group mean values for dpm/animal were: Concentration mean dpm/animal 0 (vehicle control) 734.50 10 1554.32 25 1732.96 50 2021.45

Any other information on results incl. tables

A Stimulation Index of less than 3 was recorded for the three concentrations of the test material.

No signs of systemic toxicity were noted in either the control or test animals and bodyweight changes of test animals were comparable to those of the control group. Off white resiual test material on the ears was noted post-dose on Day 3 in animals in the 10% w/w group. Off white residual test material on the ears and/or fur loss were noted in all animals in the 25% and 50% w/w groups on Day 2 and for the remainder of the test.

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:

The substance was tested for skin sensitisation potential in the mouse local lymph node assay according to OECD Test Guideline 429. Four groups of five mice were treated with the test material at concentrations of 0%, 10%, 25% and 50% w/w in butanone. The mice were treated by daily application of 25 microlitres of the appropriate concentration of test material to the dorsal surface of each ear for three consecutive days. five days following the first topical application (Day 6) all mice were injected via the tail vein with 250 microlitres of phosphate buffered saline containing 3H-methyl thymidine. Five hours later, all animals were killed by carbond dioxide asphyxiation and draining auricular lymph nodes were removed and processed.

The Stimulation Index, calculated as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 2.12 at 10%, 2.36 at 25%, and 2.75 at 50% w/w. The Stimulation Index did not exceed 3 in any treatment group; therefore, the test material was considered to be a non-sensitiser under the conditions of the test.