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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
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)
Version / remarks:
of 2002
Deviations:
yes
Remarks:
Contrary to OECD 429 of 2002, but in accordance with OECD 429 of 2010, scintillation vials were filled with 10 mL of scintillation fluid for 3H-counting. This did not compromise the validity of the study.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
of 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Mouse (healthy females, nulliparous and non-pregnant), strain: CBA/CaOlaHsd with appropriate range of bodyweight at study start.
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at treatment start (1st induction): 8 to 12 weeks.
- Weight at treatment start (1st induction): Minimum 19.0 g, maximum 22.3 g.
- Housing: Individual housing in Makrolon Type II cages with wire mesh top.
- Bedding material: Granulated softwood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany).
- Diet (ad libitum): Pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst / The Netherlands).
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days before treatment start
under the same conventional standard laboratory conditions as the LLNA was conducted.

ENVIRONMENTAL CONDITIONS

Standard laboratory conditions, environmental conditions were set at:
- Temperature (°C): 22 ± 2°C
- Relative Humidity (%): 45 to 65%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
For few hours relative humidity in the animal room was outside the target range and reached minimum and maximum records of ca. 39 and 81%, respectively. This minor deviation did not compromise the integrity or validity of the study.



Vehicle:
dimethylformamide
Concentration:
Induction on Days 1, 2 and 3 at the following concentrations of THEIC in the vehicle (w/v):
- Pre-study: 10% (1 female), 25% (1 female)
- Main Study: 0% (vehicle control, 4 females), 5% (4 females), 10% (4 females), 25% (4 females).
No. of animals per dose:
- Pre-study: 1 female animal per dose
- Main Study: 4 female animals per dose
Details on study design:
Test Substance Solubility:

A vehicle trial has demonstrated that the highest test substance concentration suitable for use in the LLNA is a 25 % (w/v) solution in dimethylformamide. At higher test substance concentrations a suitable solution or suspension was not achievable, neither by use of other vehicles suitable for use in LLNAs nor by warming to 37°C.

Treatment Preparation and Administration for the Pre-test and Main Test:

On three consecutive days, groups of 1 female mouse (pre-test) or 4 female mice (main test) were treated once daily by topical application to the entire dorsal surface of both ears with 25 μL/ear at the foreseen concentrations (w/v) of test substance in the vehicle. In the same manner, animals of the vehicle control group received the vehicle alone. Test substance preparations were newly prepared on each day of dosing.

Observations, Measurements and Endpoints (Pooled treatment group approach):

All animals were checked daily for signs of local irritation at the application site and systemic toxicity. In addition, bodyweights were recorded on Days 1 (prior to treatment) and 6 (pre-study animals prior to sacrifice, main study animals prior to injection of 3H-methyl thymidine (3HTDR)). On Day 6, all main study animals were injected into the tail vein 3HTDR diluted in phosphate buffered saline at a nominal dose of ca. 20.4 µCi per mouse, in order to measure lymphocyte proliferation by radioactive labelling. Approximately five hours afterwards the draining (auricular) lymph nodes were excised and pooled for each experimental group. After precipitating macromolecules of the lymph node cells, radioactivity measurements were performed on Day 7 on a β-scintillation counter. Radioactivity was expressed as the number of radioactive disintegrations per minute (dpm). The ratio of the proliferation (reflected by the magnitude of measured dpm/node) in treated groups to that in the vehicle control group, termed the stimulation index (SI), was subsequently calculated for each group. Mean scintillation-background DPM was duely accounted for in all groups.

Criteria Used to Consider a Positive Response:

The test substance is regarded as a sensitizer if at least one concentration of the test substance produces a stimulation index (SI) ≥ 3.
In addition, due consideration is given to dose-relationship of the attained stimulation indices, although skin penetration properties of test substances, local toxicity and/or immunosuppression may interfere with dose-relationship.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Dose response was not assessed by statistical analysis, because Stimulation Indices well below 3 were attained at all tested concentrations and there was no indication of a dose related increase in DPM/lymph node.
Positive control results:
Stimulation indices (SI) of 1.00 (vehicle control), 2.04, 3.41 and 6.14 were attained in a contemporaneous positive control assay with the same strain of mice (CBA/CaOlaHsd) in response to 0 (vehicle control), 5, 10 and 25% w/v hexyl cinnamic aldehyde in acetone:olive (4:1 v/v), respectively, thus demonstrating the reliability and sensitivity of this test system and assay to detect skin sensitization potential in this laboratory.
Key result
Parameter:
SI
Value:
1.25
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.24
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.01
Test group / Remarks:
25%

Mortality / clinical signs:

There were no deaths and no clinical signs indicative of systemic effects or of local toxicity at the ears during the main study period. During the pre-test, ruffled fur, slight in degree was recorded approximately 24 hours after the second and approximately 24 h after the third administration in the animal treated with the 25% w/v test substance solution. This minor finding had resolved by pre-test day 5 and was not considered necessitating exclusion of this test concentration from the main study.

Body weight:

There was no indication of adverse effects on bodyweight attributable to treatment with the test substance.

Interpretation of results:
other: not sensitising
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the low stimulation indices attained in the local lymph node assay, THEIC is considered not to be a skin sensitiser and does not warrant any classification regarding skin sensitisation according to European classification rules [REGULATION (EC) 1272/2008].