Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other 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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): XU-18838.00
- Physical state: solid
- Lot/batch No.: 20120135-18
- Expiration date of the lot/batch: June 18, 2014
- Stability under test conditions: Test substance was expected to be stable for the duration of testing
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Inc., Frederick, MD
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 18.5-23.3 grams
- Housing: individually housed in plastic solid bottom cages with
bedding during the dosing and resting phases of the study.
- Diet (e.g. ad libitum): Harlan Teklad Certified Global 16% Protein Rodent Diet® #2016C. The diet
was available ad libitum.
- Water (e.g. ad libitum): Filtered tap water was supplied ad libitum by an automatic water dispensing
system.
- Acclimation period: 7-21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-27ºC
- Humidity (%): 45-62%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: May 1 - June 14, 2012

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
5% 10% and 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: test material was soluble in Methyl Ethyl Ketone and could be dosed in the
LLNA at a maximum concentration of 25% based on handling and dosing considerations.
- Irritation: No dermal irritation was observed at any vehicle control site or any treated site
dosed with 5% and 10% of test substance.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Proper
conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact
sensitizer, which did elicit a proliferative response with a SI value of 3.48 relative to vehicle
controls.

TREATMENT PREPARATION AND ADMINISTRATION:
Dilutions of the test substance were prepared as w/w mixtures in Methyl Ethyl Ketone.
Concentrations of 5%, 10% and 25% were selected for the main test based on results of the
preliminary screening test. A single concentration of a 25% w/w mixture of HCA in Methyl
Ethyl Ketone was also prepared as a positive control. All dosage preparations were freshly
prepared on the day of administration.
Beginning on Day 1, a volume of 25 L of the appropriate test substance concentration, the
positive control substance, or the vehicle was applied to the dorsum of both ears of each mouse
once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During
application, the material was gently spread as evenly as possible 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:
Statistical analysis was performed on the dpm, body weight and body weight gain values.
Significance was judged at p <0.05. The treated groups and 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) (INSTAT
Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs
test (1969).

Proper
conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact
sensitizer, which did elicit a proliferative response with a SI value of 3.48 relative to vehicle
controls.

Results and discussion

Positive control results:
Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer, which did elicit a proliferative
response with a SI value of 3.48 relative to vehicle controls.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 5% Test Substance in Methyl Ethyl Ketone - 1.70 10% Test Substance in Methyl Ethyl Ketone - 0.90 25% Test Substance in Methyl Ethyl Ketone - 2.08 Positive Control (25% HCA in Methyl Ethyl Ketone) - 3.48
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Vehicle Control (Methyl Ethyl Ketone) - 1059.97 ± 145.39 5% Test Substance in Methyl Ethyl Ketone - 1805.61 ± 558.71 10% Test Substance in Methyl Ethyl Ketone - 954.58 ± 447.86 25% Test Substance in Methyl Ethyl Ketone - 2205.47 ± 1049.95 Positive Control (25% HCA in Methyl Ethyl Ketone) - 3685.87 ± 1013.93

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: UN GHS
Conclusions:
Based on the results of this study, the test substance is considered to lack dermal sensitization
potential in the LLNA at concentrations of less than or equal to 25%. Proper conduct of the
LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.
Executive summary:

A dermal sensitization test was conducted with female mice to determine the potential for XU-18838.00 to produce sensitization after repeated topical applications. Preliminary solubility and handling evaluations indicated the test material was soluble in Methyl Ethyl Ketone and could be dosed in the LLNA at a maximum concentration of 25% based on handling and dosing considerations. During the initial test, the positive control (25% HCA in Methyl Ethyl Ketone) failed to elicit a positive response, therefore, the test was repeated to achieve a valid positive control result. The body of this report reflects the second test using the same procedure of the initial test. The DPM values for the initial test are presented in Appendix C (see Section 11). Three concentrations (5%, 10% and 25%) of the test substance in Methyl Ethyl Ketone or the vehicle alone were topically applied to twenty healthy female test mice (5 mice/group) 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 in disintegrations per minute per mouse (dpm/mouse). Each animal’s ears were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the IV injection. A positive control group (five female mice) was maintained under the same environmental conditions and treated with a 25% w/w mixture of alpha-Hexylcinnamaldehyde Technical (HCA) in Methyl Ethyl Ketone in the same manner as the test animals.

Based on the results of this study, the test substance is considered to lack dermal sensitization potential in the LLNA at concentrations of less than or equal to 25%. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer. 1