Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 February, 2015 - 23 March, 2015
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:
(July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(May 2008, including most recent amendments)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(March 2003)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): NS-1000
- Appearance: Clear colourless liquid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 18 - 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 (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 1, 2 and 5%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Initially, two test substance concentrations were tested; a 100% and 50% concentration. The highest concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11-12 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.
Based on the results of the initially treated animals, eight additional animals were treated in a similar manner with four lower concentrations (25%, 10%, 5% and 2%) at a later stage.

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 using the individual SI values. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

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 preparations (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 on the basis of maximizing the solubility using the test substance data provided by the sponsor and trial preparation results performed at WIL Research Europe.

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 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy for gross macroscopic examination was performed according to protocol.
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 (see attached reliability check) indicates that the Local Lymph Node Assay as performed at WIl Research Europe is an appropriate 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 1, 2 and 5% were 1.7, 1.1 and 0.8, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 1, 2 and 5% were 1413, 953 and 665 DPM, respectively. The mean DPM/animal value for the vehicle control group was 856 DPM.

Any other information on results incl. tables

Results Pre-screen test:

Lethargy, hunched posture and/or piloerection were noted for the animals treated at 50% and 100% between Days 1 and 3. Hunched posture and piloerection were also noted for the animals treated at 10% and 25% on Days 1 and 2. These animals were sacrificed for humane reasons on Day 2. Marked body weight loss was observed for individual animals treated at 100%, 50%, 25% and 10%. No signs of toxicity were observed in the animals treated at 5% and 2%. No irritation was observed in any of the animals examined and 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 5% concentration.

Other results - main study:

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.

 

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 429 test guideline and GLP principles, NS-1000 was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to and including 5% v/v.
Executive summary:

An LLNA skin sensitisation study was performed with NS-1000 according to OECD 429 test guideline and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 1%, 2% and 5% v/v. 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. 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. 

Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 1413, 953 and 665 DPM, respectively. The mean DPM/animal value for the vehicle control group was 856 DPM. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.7, 1.1 and 0.8, respectively. As the SI appeared not to be ≥ 3 when tested up to 5% v/v, NS-1000 was considered not to be a skin sensitiser.

Based on the results of this study, the substance does not need to be classified for skin sensitisation according to Regulation (EC) No 1272/2008 on classification, labelling and packaging.