Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 15, 2014 - November 10, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): L 130
- Appearance: Faint light-green, powdery solid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 20-23 g
- Housing: Animals were group housed in labeled Makrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: October 15, 2014 - November 10, 2014

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0, 10, 25, 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
In a pre-screen test, two test substance concentrations were tested, a 25% and 50% concentration. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
INTERPRETATION
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
- Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research.

Performed according to test guidelines:
- Days 1, 2 and 3: Induction (topical treatment of 25 µL/ear)
- Day 6: Injection of 20 µCi 3H-methyl thymidine. Five hours after the injection, all animals were killed and the ear lymph nodes were excised. A cell suspension of lymph nodes was prepared.
- Day 7: Radioactivity measurements

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 within 1 hour after dosing)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity. The SI values calculated for the concentrations 5, 10 and 25% were 1.1, 3.1 and 3.7, respectively. An EC3 value of 9.8% was calculated using linear interpolation.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.3, 1.1 and 1.2, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1080, 887 and 978 DPM, respectively. The mean DPM/animal value for the vehicle control group was 802 DPM.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. White staining of test substance remnants on the dorsal surface of the ears of both animals treated at 50% on Days 1, 2 and 3, did not hamper scoring of erythema. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined. White staining of test substance remnants on the dorsal surface of the ears of all animals treated at 50% on Days 1, 2 and 3 did not hamper scoring of erythema.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

No mortality occurred and no clinical signs of systemic toxicity were observed. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In a LLNA skin sensitisation study, performed according to OECD/EC test guidelines, L 130 (a solid) was considered not be a skin sensitiser, as the SI was shown to be < 3 when tested up to and including 50%.
Executive summary:

A LLNA skin sensitisation study was performed according to OECD/EC test guidelines with L 130. Based on the results of a pre-screen test, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (propylene glycol). No irritation of the ears was observed in any of the animals examined. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1080, 887 and 978 DPM, respectively. The mean DPM/animal value for the vehicle control group was 802 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.3, 1.1 and 1.2, respectively. Based on these data, the substance is considered not be a skin sensitiser, as the SI was shown to be < 3 when tested up to and including 50%.