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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Isodur
- Physical state: solid
- Analytical purity: 92.7 %
- Stability under test conditions: According to the sponsor, the stability of the test substance was guaranteed/ verified throughout the study period.
- Storage condition of test material: ambient temperature

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Laboratories
- Age at study initiation: 6 – 12 weeks
- Weight at study initiation: 18.2 g – 21.3 g
- Housing: single housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

- Temperature (°C): 20 – 24°C
- Humidity (%): 30 – 70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Study design: in vivo (LLNA)

propylene glycol
50% in propylene glycol
No. of animals per dose:
5 females
Details on study design:
- Compound solubility: The 50% preparation was the maximum technically applicable concentration.
- Irritation: very slight irritation was observed in the range finding test at a 50 % concentration but not in the main study at the same concentration
- Lymph node proliferation response: no biologically relevant response was observed

- Name of test method: LLNA according to OECD 429 guideline
- Criteria used to consider a positive response:
In order to reveal a possible induction of sensitization, the response in the draining lymph
node after epicutaneous application of several concentrations of the test substance to the
skin of the ear backs is determined. The parameters used to characterize the response are
lymph node cell count, 3H-thymidine incorporation into the lymph node cells and to a certain
extent lymph node weight. Because not only sensitization induction but also irritation of the
ear skin by the test substance may induce lymph node responses, the weight of ear punches
taken from the area of test-substance application is determined as a parameter for
inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
The increase SI of cell count by a factor of ≥ 1.5 and/or of 3H-thymidine incorporation by a
factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as
indicating a sensitizing potential of a test substance.
If applicable, the EC (estimated concentration) leading to the respective SI values were
calculated by linear or semi-logarithmical regression between the data points directly below
and above the SI if possible or using the two nearest points below or above the SI.
In addition the evaluation uses the following considerations:
If biologically relevant increases in ear weights are running in parallel to the increase in cell
count, 3H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the
lymph node response was caused by irritation and not by skin sensitization. Depending on
the magnitude of lymph node response the evaluation of the sensitizing potential may be
modified or additional studies might be necessary by expert judgement.
If a test substance does not elicit a biological relevant increase in cell count, 3H-thymidine
incorporation but shows a clear concentration related increase in response, further
investigation of the sensitization potential at higher concentrations should be considered.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
not applicable

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Test group / Remarks:
Remarks on result:
other: see Remark
When applied as 50% preparation in propylene glycol, the test substance did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no increase in lymph node weights, as well.
other: disintegrations per minute (DPM)
Remarks on result:
other: vehicle control (propylene glycol): 418.3 dpm/lymphnode pair 50% in propylene glycol: 657.8 dpm/lymphnode pair

Any other information on results incl. tables

The skin sensitizing potential of Isodur was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/J mice each were treated with a 50% w/w preparation of the test substance in propylene glycol or with the vehicle alone. The 50% preparation was the maximum technically applicable concentration. The study used 1 test group and 1 control group. Each test animal was applied with 25 μL per ear of the test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μL of sterile saline into a tail vein. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and 3H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each test group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed. When applied as 50% preparation in propylene glycol, the test substance did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no increase in lymph node weights, as well. Concomitantly, the increase of 3H-thymidine incorporation into the cells was not biologically relevant (no increase above the cut off stimulation index of 3).

The test-substance preparation did not cause any increase in ear weights. Thus it is concluded that Isodur does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Applicant's summary and conclusion