Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 December 2011 - 16 January 2012
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 (incl. certificate)
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): FAT 40854/A TE
- Substance type: Reddish-brown powder
- Physical state:solid
- Purity: 46.2% (4 main constituents)
- Lot/batch No.: TZ 5719 / BOP 02-11
- Expiration date of the lot/batch: 01 April 2016
- Storage condition of test material: At room temperature in the dark
- Hygroscopic: Yes, store in well-sealed container
- Volatile: No
- pH: 5.4 at concentration of >80g/L

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 labeld 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): 17.9 – 20.7ºC
- Humidity (%): 41 – 72%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Deviations from the minimum level of temperature occurred. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 14 December 2011 - 16 January 2012

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10, 25, 50%
No. of animals per dose:
5
Details on study design:
The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity).

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. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (20011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.

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 treatment. 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 following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: All animals surviving to the end of the study were sacrificed by intra-peritoneal injection with Euthasol® 20% (0.2 mL/animal).
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 model for testing for contact hypersensitivity. See attached document 'Reliability check'.

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 11.7, 13.4 and 20.1 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 5481, 6308 and 9451 DPM respectively. The mean DPM/animal value for the vehicle control group was 470 DPM.

Any other information on results incl. tables

Results Pre-screen test:

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Red test substance remnants prevented scoring for erythema, except for scoring prior to dosing on Day 2. At that observation no erythema was noted in all animals at 25 and 50%. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Other results - main study:

Skin reactions / Irritation:

Red test substance remnants prevented scoring for erythema.

Systemic toxicity/Body weights:

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.

Macroscopy of the auricular lymph nodes and surrounding area:

All auricular lymph nodes of the animals of the experimental groups were considered larger in size compared to normal. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Conclusions:
The SI values calculated for the substance concentrations 10, 25 and 50% were 11.7, 13.4 and 20.1 respectively.
These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be lower than10%.
Executive summary:

In an LLNA study performed according to OECD 429, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 10, 25 and 50% or dimethylformamide on three consecutive days by open application on the ears. Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after 5 hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. The positive control showed appropriate results.

The SI values calculated for the substance concentrations 10, 25 and 50% were 11.7, 13.4 and 20.1, respectively. The EC3 value was established to be lower than 10%. FAT 40854/A TE is considered to be a skin sensitiser.