Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April, 2015 - 11 May, 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 lymphnode 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): IN-1000
- Appearance: Dark amber liquid
- Storage condition of test material: In refrigerator (2-8°C) container flushed with nitrogen

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: 20 - 24 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, 2, 5 and 10%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Initially, two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).
The test system, procedures and techniques were identical as 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. The animals were sacrificed after the final observation.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (10% and 5%) at a later stage. Based on the results of the these animals, two additional animals were treated in a similar manner at 25% 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 the DPM/vehicle control group mean.
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 correction for the purity of the test susbtance and 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.

Necropsy: All moribund animals were sacrificed by intra-peritoneal injection with Euthasol® 20% (0.2 mL/animal). Animals found dead or sacrificed for humane reasons were subjected to necropsy for gross macroscopic examination.
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 document) 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 2% and 5% were 1.4 and 1.4.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 2% and 5% were 1066 and 1076 DPM, respectively. The mean DPM/animal value for the vehicle control group was 772 DPM.

Any other information on results incl. tables

Results Pre-screen test:

At 50% and 100%, hunched posture was noted for all animals on Day 2. One animal treated at 50% was found dead on Day 3. Hunched posture, piloerection, lethargy and hypothermia were noted for the surviving animals on Day 3. The other initially treated animals were sacrificed for humane reasons on Day 3.

At 5% and 10%, no systemic toxicity was observed.

At 25%, hunched posture was noted for one animal on Day 1. On Day 2 this animal also showed piloerection. Hunched posture was also noted for the other animal on Day 2. Both animals were sacrificed for humane reasons on Day 2.

No irritation was observed in any of the animals examined. 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 10% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined.

 

At 10%, one animal was found dead on Day 2. Three animals showed hunched posture and piloerection on Day 2. All animals of this group were sacrificed for humane reasons on Day 2. No abnormalities were found at macroscopic post mortem examination of these animals.

At 5%, one animal showed piloerection on Day 3 and hunched posture between Days 4 and 6. Additionally, this animal showed body weight loss. Since the systemic toxicity, shown by this animal, can interfere with the endpoint of the study, the animal was excluded from interpretation.

In the other animals treated at 5% and the animals treated at 2%, no mortality occurred and no clinical signs of systemic toxicity were observed. Body weights and body weight gain remained in the same range as controls over the study period.

Based on the available data, it was considered that the study outcome was not adversely affected by the loss of the animals treated at 10% and animal treated at 5%.

 

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.

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, IN-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:

A LLNA skin sensitisation study was performed with IN-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 2%, 5% and 10% v/v. Reliable negative and positive controls were included.

No irritation of the ears was observed in any of the animals examined.

At 10%, one animal was found dead on Day 2. Three animals showed hunched posture and piloerection on Day 2. All animals of this group were sacrificed for humane reasons on Day 2. No abnormalities were found at macroscopic post mortem examination of these animals.

At 5%, one animal showed piloerection on Day 3 and hunched posture between Days 4 and 6. Additionally, this animal showed body weight loss. Since the systemic toxicity, shown by this animal, can interfere with the endpoint of the study, the animal was excluded from interpretation.

In the other animals treated at 5% and the animals treated at 2%, no mortality occurred and no clinical signs of systemic toxicity were observed. Body weights and body weight gain remained in the same range as controls over the study period.

Based on the available data, it was considered that the study outcome was not adversely affected by the loss of the animals treated at 10% and animal treated at 5%. 

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 item concentrations 2 and 5% were 1066 and 1076 DPM, respectively. The mean DPM/animal value for the vehicle control group was 772 DPM. The SI values calculated for the test item concentrations 2 and 5% were 1.4 and 1.4, respectively. As the SI appeared not to be ≥ 3 when tested up to 5% v/v, IN-1000 was considered not to be a skin sensitiser and the substance does not need to be classified for skin sensitisation according to Regulation (EC) No 1272/2008 on classification, labelling and packaging.