Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 November - 12 December2011
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 lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 41043/A TE
- Substance type: Red powder, solid
- Physical state: Powder
- Purity: 99.8%
- Lot/batch No.: DRI 2398 Op. 4 stabilisiert
- Expiration date of the lot/batch: 30 April 2016
- Storage condition of test material: At room temperature in the dark
- Hygroscopic: No
- pH: 5.54 of a solution/dispersion of 10% (w/w)

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: Body weight variation was within +/- 20% of the sex mean (range: 22-26 grams).
- 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): 18.1 - 22.6
- Humidity (%): 44 - 68
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 November - 12 December 2011

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0, 5, 10, 25%
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:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2 (see section 6.7) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 10% and 25% concentration. The highest concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). 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 prior to dosing on Days 1 and 3, and on Day 6.
Since test substance remnants were anticipated to hamper scoring after dosing on Days 2 and 3 (based on Day 1 observations) and to hamper ear thickness measurements on Days 3 and 6, the ears were cleaned of residual test substance with tap water and the selected vehicle on Days 2, 3 and 6 between 30 and 60 minutes prior to any further assessment. An additional scoring of the ears was conducted prior to dosing on Days 2 and 3, next to scoring of the ears after dosing. Ear thickness measurements on Days 3 and 6 were conducted following the scoring of irritation and prior to dosing (if applicable). Animals were sacrificed after the final observation. No necropsy was conducted on the animals of the pre-screen test.

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 (2007) 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. Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3)

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

The result of the macroscopic examination of the auricular lymph nodes and surrounding area of animal nos. 6 and 11 was not recorded (except for relative size of the lymph nodes). Sufficient information was available for adequate interpretation of the study results.

Necropsy: After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
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 5, 10 and 25% were 2.2, 1.7 and 1.8, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 143, 105 and 114 DPM, respectively. The mean DPM/animal value for the vehicle control group was 64 DPM.

Any other information on results incl. tables

Results Pre-screen test:

No signs of systemic toxicity were 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. Purple/red test substance remnants were present on the dorsal surface of the ears of all animals at 10 and 25% throughout the observation period, which prevented scoring for erythema.

Based on the results, the highest test substance concentration selected for the main study was a 25% concentration.

Other results - main study:

Skin reactions / Irritation (see Table 3)

Purple/red test substance remnants were present on the dorsal surface of the ears of all animals at 5, 10 and 25% throughout the observation period, which prevented scoring for erythema.

Systemic toxicity (Body weights, see Table 3)

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. The body weight loss noted for several animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

Macroscopy of the auricular lymph nodes and surrounding area (Table 4)

The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal at 5% and three animals at 25%, which appeared larger in size when compared to the other treated groups. No macroscopic abnormalities of the surrounding area were noted in 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 a LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered not be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 25%.

The SI values calculated for the substance concentrations 5, 10 and 25% were 2.2, 1.7 and 1.8 respectively (Table 4 and Figure 1).
Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 25%, FAT 41043/A TE was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

Based on these results, FAT 41043/A TE would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.
Executive summary:

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5, 10 or 25% 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). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five 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 SI values calculated for the substance concentrations 5, 10 and 25% were 2.2, 1.7 and 1.8 respectively.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 25%, FAT 41043/A TE was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

Based on these results, FAT 41043/A TE would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.