Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-02-29 to 2012-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: 100% UVCB
- Physical state: Dark coloured liquid
- Expiration date of the lot/batch: February 2, 2013
- Stability under test conditions: stable for duration of testing
- Other: Solubility in various solvents unknown

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Inc.
- Age at study initiation: 11 weeks
- Weight at study initiation: 19.2 - 25.4 g
- Housing: Plastic solid bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8-21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26°C
- Humidity (%): 45-53%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 2012-02-29 To: 2012-040-04

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: 80, 50, 25, 20, 10 and 5%
Main test: 10, 5 and 1%
No. of animals per dose:
5/dose level
Details on study design:
RANGE FINDING TESTS:
- Irritation: Scored for erythema pre-dose Days 1, 2, 3 and on Day 6. Ear-swelling measurements (duplicate) of each animal on Day 1 (pre-dose), Day 3 (ca. 48 hours after 1st dose) and Day 6 prior to scarifice.
- Lymph node proliferation response: Not measured

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Random assignment
- Criteria used to consider a positive response: Erythema and Oedema to modified Draize scoring method. Ear- swelling based on percentage change from control. Sensitisation respnse by B-scintillation (disintegrations per minute, dpm); mean dpm per test group /mean dpm of vehicle control group. SI > 3 = positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
Dilutions of the test substance were prepared as w/w mixtures in acetone/olive oil (AOO 4:1 v/v). A single concentration of the positive control (25%w/w mixture of HCA in AOO) was prepared. All dosage preparations were freshly prepared on the day of administration.
Beginning on Day 1, 25µL of the appropriate test substance concentration, vehicle alone or the positive control substance was applied tothe dorsum of bpth ears of each mouse once per day for three consecutive days using a micropipette. During application, the material was spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.
On Day 6 of the study ( 3 days after the final topical application) 250 µL of sterile phosphate buffered saline containing 20 µCi of 3-H-methyl thymidine was injected intravenously via the tail vein of each mouse.
Approximately 5 hours after the injection, the draining auricular lymph nodes from all animals were excised. The lymph nodes were pooled for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of two microscope slides ovaer a collection vessel. The slides were then briefly rinsed with PBS into the vessel. The vessel contents were centrifuged for approximately 10 minues at 1800 rpm twice and washed. After the second wash ca. 5 mL of 5% trichloracetic acid (TCA) in distilled water was added to the sediment and vortexed. The DNA was precipitated in the 5% TCA at 4.0 - 5.2°C overnight (ca. 18 hours).
After precipitation the tubes were centrifuged and the suprnatent discarded. The resulting precipitate was resuspended in 1 mL of 5% TCA and transferred to 10 mL scintillation fluid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Significance judged at p<0.05. Treated groups and negative vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett's t-test for multiple comparisons. Where variances are considered significantly different by Bartlett's test groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn's test). Outlier analysis was conducted using Grubbs (1969).

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
See Table 4 in attached document: LLNA_Durando 2012_Results Tables Treatment of mice with 1, 5 or 10% of the test material resulted in stimulation index values of 0.95, 2.07 and 3.96, respectively, relative to vehicle control mice. The substance was considered positive for a dermal sensitisation potential with a score > 3 at a test concentration at 10%. The positive control elicited a response of 4.66 relative to the vehicle control. The EC3 values was calculated to be 7%.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 3 in attached document: LLNA_Durando 2012_Results Tables

Any other information on results incl. tables

Although nine mice lost or failed to gain body weight during the study, all animals appeared active and healthy throughout the study.

Group 1; Vehicle control: Very slight erythema noted at two test sites between Days 2 and/or 3

Group 2; Positive control: Very slight erythema noted at most sites between Days 2 and/or 6

Group 3; 1% test conc.: Very slight erythema noted at three sites between Days 2 and/or 6

Group 4; 5% test conc.: Very slight erythema noted at most sites between Days 2 and/or 6

Group 5; 10% test conc.: Very slight to well-defined erythema noted at all sites between Days 2 and/or 6

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance is considered to be a contact dermal sensitiser at concentrations of greater than or equal to 7% (EC3 value)
Executive summary:

Test Guidance

OECD 429, EU Method B42 and US EPA OPPTS 870.2600

Method and material

Three concentrations of the test substance in acetone/olive oil (AOO 4:1 v/v) or the vehicle alone were topically applied to twenty healthy mice (5/dose) for three consecutive days. Three days after the last application, the mice were given a 20 µCi IV injection of 3H-methyl thymidine. Five hours later, the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter. The results are presented as disintegrations per minutes per mouse (dpm/mouse). Each animals ears were also evaluated for erythema and oedema prior to each application and again on Day 6, prior to the IV injection. A positive control group (5 animals) was maintained under the same environmental conditions and treated with 25% w/w mixture of alpha-hexylcinnamaldehyde (HCA) in AOO in the same manner as the test animals. Ear thickness (swelling) was taken on the preliminary, test and control animals on Day 1 (predose), Day 3 (ca 48 hours aftre the first dose), and Day 6 prior to sacrifice.

Results

Treatment of mice with 1, 5 or 10% of the test material resulted in stimulation index values of 0.95, 2.07 and 3.96, respectively, relative to vehicle control mice. The substance was considered positive for a dermal sensitisation potential with a score > 3 at a test concentration at 10%. The positive control elicited a response of 4.66 relative to the vehicle control. The EC3 values was calculated to be 7%.

Conclusions

The test substance is considered to be a contact dermal sensitiser at concentrations of greater than or equal to 7% (EC3 value)