Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14NOV2012 to 03DEC2012
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.
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)
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 females (approx. 10 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- 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
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14NOV2012 to 03DEC2012
Vehicle:
propylene glycol
Concentration:
5, 10, 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two test substance concentrations were tested; a 50% and 25% concentration. 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. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

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.

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. In order to obtain homogeneity, the test substance (formulations) were heated in a water bath with a maximum temperature of 51.1ºC for a maximum of 0.5 hours. Temperature of the water bath used for heating the formulation to be used for the first and third induction of the main study exceeded the maximum allowed temperature of 50°C with 0.6 and 1.1°C, respectively.The deviation was very slight in nature, and information provided by the sponsor indicated that the test substance is stable at 50°C. This deviation was therefore considered not to have adversely affected the integrity of the test substance. The test substance (formulations) were allowed to cool down to a temperature below 40 ºC prior to dosing.

Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe 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). Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: All moribund animals were sacrificed by intra-peritoneal injection with Euthasol® 20% (0.2 mL/animal). No necropsy was conducted on the animals that were found dead or sacrificed for humane reasons.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 5 and 10% were 0.5 and 1.1 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM/animal values for the experimental groups treated with test substance concentrations 5 and 10% were 206 and 481 DPM respectively. The mean DPM/animal value for the vehicle control group was 425. Data obtained from animals treated with a 25% test substance concentration were not used for interpretation due to occurrence of systemic toxicity.

Results Pre-screen test:

Both animals at a 50% test substance concentration were sacrificed for ethical reasons or found dead on Day 2. Clinical signs noted for the sacrificed animal consisted of hunched posture, piloerection, lethargy and ptosis. Slight erythema was noted on both ears of these animals on Day 2. No signs of irritation were noted at a 25% test substance concentration. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values (no measurements were done in both animals at 50% on Days 3 and 6). 

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

Other results - main study:

Two animals at 25% were sacrificed for ethical reasons on Days 3 or 4, respectively. Clinical signs noted for both of these animals included flat posture, ptosis, tremors and/or irregular breathing on Day 3. One of the surviving animal showed tremors and abnormal gait on Day 3.

 

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

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

The auricular lymph nodes of all (surviving) animals treated with a 10% and 25% test substance concentration appeared larger in size when compared to the other treated groups. All auricular lymph nodes of the animals of the control animals and animals at a 5% test substance concentration were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the surviving animals.

 

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 considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 10%.
Executive summary:

An LLNA skin sensitisation assay was performed according to OECD/EC guidelines and GLP principles. The test substance was applied at concentrations of 5, 10 or 25%, however two of the three animals in the highest exposure group had to be sacrificed due to severe systemic toxicity. Data obtained at this concentration were not used for interpretation.

In the other groups, no significant body weight loss was noted, and no irritation of the ears was observed. The auricular lymph nodes of all (surviving) animals treated with a 10% and 25% test substance concentration appeared larger in size when compared to the other treated groups, the auricular lymph nodes of animals at 5% test substance concentration were considered normal in size. The SI values calculated for the substance concentrations 5 and 10% were 0.5 and 1.1 respectively.

Based on these data, TMAC is considered not to be a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

An LLNA skin sensitisation assay was performed according to OECD/EC guidelines and GLP principles. The test substance was applied at concentrations of 5, 10 or 25%, however, two of the three animals in the highest exposure group had to be sacrificed due to severe systemic toxicity (flat posture, ptosis, tremors and/or irregular breathing). Data obtained at this concentration were not used for interpretation.

In the other groups, no significant body weight loss was noted, and no irritation of the ears was observed. The auricular lymph nodes of all (surviving) animals treated with a 10% and 25% test substance concentration appeared larger in size when compared to the other treated groups, the auricular lymph nodes of animals at 5% test substance concentration were considered normal in size. The SI values calculated for the substance concentrations 5 and 10% were 0.5 and 1.1 respectively.


Migrated from Short description of key information:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 10%.

Justification for selection of skin sensitisation endpoint:
One study available. Study performed according to OECD guideline and GLP principles (reliability 1).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, TMAC is not classified for skin sensitization according to CLP Regulation (EC) No. 1272/2008.