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Description of key information

In an LLNA study with mice, SI values calculated for the concentrations of 0.5, 1 and 2.5% the test substance were 0.9, 1.2 and 2.2, respectively. These results indicate that the test substance did not elicit an SI ≥ 3. The substance is therefore not considered to be sensitizing.

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:
From 04 April, 2012 to 07 May, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 animals (approx 11 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 d

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approx 15 room air changes/h, and a 12-h light/12-h dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Deviations from the minimum level of daily mean relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 04 April - 07 May 2012
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test concentrations: 0, 0.5, 1, 2.5%
Pre-screen test was done at doses of 0.5, 1, 2.5, 5, 10, 50 and 100%. Up to doses of 5%, ear swelling and signs of erythema were too pronounced. In the OECD TG 429, it is written “excessive local skin irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement. The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series that does not induce systemic toxicity and/or excessive local skin irritation.“. In the case of di-TMPTTA, the threshold of 25% of ear thickness was exceeded at the doses of 5% and higher. Therefore the main study has been conducted with doses of 0.5, 1 and 2.5%.
No. of animals per dose:
5
Details on study design:
The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
Pre-screen test was done at doses of 0.5, 1, 2.5, 5, 10, 50 and 100%. Up to doses of 5%, ear swelling and signs of erythema were too pronounced. In the OECD TG 429, it is written “excessive local skin irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement. The highest dose selected for the main LLNA study will be the next lower dose in the pre-screen concentration series that does not induce systemic toxicity and/or excessive local skin irritation.“. In the case of di-TMPTTA, the threshold of 25% of ear thickness was exceeded at the doses of 5% and higher. Therefore the main study has been conducted with doses of 0.5, 1 and 2.5%.

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 h prior to each treatment. Homogeneity was obtained to visually acceptable levels.
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 h 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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. See attached document 'Reliability check'.
Key result
Parameter:
SI
Value:
ca. 0.9
Variability:
five animals
Test group / Remarks:
0.5% test substance
Parameter:
other: disintegrations per minute (DPM)
Value:
ca. 448 - ca. 1 174
Variability:
Mean DPM/animal values
Test group / Remarks:
0.5, 1 and 2.5% test substance
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.5, 1 and 2.5% were 448, 613 and 1174 DPM respectively. The mean DPM/animal value for the vehicle control group was 524 DPM.
Key result
Parameter:
SI
Value:
ca. 1.2
Variability:
five animals
Test group / Remarks:
1% test substance
Key result
Parameter:
SI
Value:
ca. 2.2
Variability:
five animals
Test group / Remarks:
2.5% test substance

Results pre-screen test:

Based on the results obtained in this test, the highest test substance concentration selected for the main study was a 2.5% concentration

Other results - main study:

Skin reactions / Irritation:

The slight irritation of the ears as shown by all animals treated at 2.5% (on Days 2 and 3) was considered not to have a toxicologically significant effect on the activity of the nodes. No irritation was shown by the other animals.

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. The body weight loss noted 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:

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 in any of the animals.

Interpretation of results:
other: CLP criteria not met
Conclusions:
In a mouse LLNA study, SI values calculated for the concentrations of 0.5, 1 and 2.5% di-TMPTTA were 0.9, 1.2 and 2.2, respectively. These results indicate that the test substance did not elicit an SI ≥ 3. The substance is therefore not considered to be sensitizing.
Executive summary:

A study was conducted to assess the skin sensitizing potential of di-TMPTTA in a mouse local lymph node assay conducted according to OECD Guideline 429, EU Method B.42 and EPA OPPTS 870.2600, in compliance with GLP. Five animals per dose were exposed to the test substance at 0.5, 1.0 or 2.5%. The slight irritation of the ears observed on Days 2 and 3 in all animals treated at 2.5% was not considered to have a toxicologically significant effect on the activity of the nodes. No irritation was shown by the other animals. 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. Mean degradations per minute (dpm)/animal at 0.5, 1 and 2.5% were 448, 613 and 1,174, respectively. The mean dpm/animal for the vehicle control group was 524. The SI values calculated for the substance concentrations of 0.5, 1.0 and 2.5% were 0.9, 1.2 and 2.2, respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 2.5%, it was established that the EC3 value (the estimated test substance concentration that will give a SI =3) exceeds 2.5%. The substance was therefore not considered to be sensitizing (Beerens 2013).

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

A study was conducted to assess the skin sensitizing potential of the test substance in a mouse local lymph node assay conducted according to OECD Guideline 429, EU Method B.42 and EPA OPPTS 870.2600. Pre-screen test was done at doses of 0.5, 1, 2.5, 5, 10, 50 and 100%. Up to doses of 5%, ear swelling and signs of erythema were too pronounced. Therefore the main study was conducted with doses of 0.5, 1 and 2.5% using five animals per dose. In this study, there was slight irritation of the ears observed on Days 2 and 3 in all animals treated at 2.5%, which was not considered to have a toxicologically significant effect on the activity of the nodes. No irritation was shown by the other animals. All auricular lymph nodes of the animals of the experimental and control groups were found to be normal in size. No macroscopic abnormalities of the surrounding area were noted. Mean degradations per minute (dpm)/animal at 0.5, 1 and 2.5% were 448, 613 and 1,174, respectively. The mean dpm/animal for the vehicle control group was 524. The SI values calculated for the substance concentrations of 0.5, 1.0 and 2.5% were 0.9, 1.2 and 2.2, respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 2.5%, it was established that the EC3 value (the estimated test substance concentration that will give a SI =3) exceeds 2.5%. The substance was therefore not considered to be sensitizing (Beerens, 2013).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No skin sensitization potential was observed up to the highest tested dose of 2.5% in a mouse local lymph node assay conducted with the test substance. Hence, the substance does not qualify for skin sensitization classification according CLP (EC 1272/2008) criteria.