Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28Sept 2011 to 17Oct 2011
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:
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:
EU Method B.42 (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:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): L579
- Substance type: white powder

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. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in 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): 19.8-23.4
- Humidity (%): 41 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 05Oct 2011 to 13Nov 2011

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0, 10, 25, 50% w/w
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
In a pre-screen test, two young adult animals per concentration (25% and 50% w/w) were treated and ear thickness was measured. Signs of systemic toxicity/ irritation and ear thickning were recorded.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- 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.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior
to each treatment. 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 NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

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 immediately after dosing) according to the numerical scoring system (see test guidelines). Furthermore, a description of all other (local) effects was recorded.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate and reliable model for testing for contact hypersensitivity.

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 228, 199 and 203 DPM respectively. The mean DPM/animal value for the vehicle control group was 175 DPM

Any other information on results incl. tables

Results Pre-screen test:

At both 25% and 50% test substance concentrations no signs of systemic toxicity were noted and no severe irritation was observed. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Test substance remnants were present on the dorsal surface of the ears of all animals on Days 1-3, but did not hamper scoring of erythema. 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. Test substance remnants were present on the dorsal surface of the ears of all treated animals on Day 1-3, but did not hamper scoring of erythema.

 

The majority of auricular lymph nodes were considered normal in size, except for the nodes of the animals treated at 10% which appeared smaller in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. 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 an LLNA skin sensitisation study performed according to OECD/EC test guidelines, the substance was found not to be a skin sensitizer, as the SI measured was not ≥3 when tested up to 50% w/w.
Executive summary:

In the mouse (local lymph node assay) contact hypersensitivity to L579 was assessed by open application of the substance (in concentrations 0, 10, 25 and 50% w/w in propylene glycol) on three sequential days to the outer ear of mice (5/ dose). On day 6, mice were injected with 3H-methyl thymidine. After 5 hours, draining lymph nodes of the ear were collected and tested for radioactivity. The SI was calculated by the ratio of the DPM/group compared to DPM/vehicle control group. No irritation of the ears was observed in any of the animals examined. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.3, 1.1 and 1.2 respectively. Since the test substance did not elicit an SI ≥ 3 when tested up to 50% w/w, L579 was considered not to be a skin sensitizer.