Morpholine, 4-[(triethoxysilyl)methyl]-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-05-18 to 2005-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD test under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-370-1

EC Name:

-

Cas Number:

21743-27-1

Molecular formula:

Hill formula: C11H25NO4Si CAS formula: C11H25NO4Si

IUPAC Name:

4-[(triethoxysilyl)methyl]morpholine

Details on test material:

- Name of test material (as cited in study report): SLM 449028
- Substance type: pure active substance
- Physical state: liquid
- Stability under test conditions: pure: stable until 2006-03-08
in water: rapid hydrolysis in water
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: 7 – 12 weeks
- Weight at study initiation: 18 - 20 g
- Housing: semi-barrier in an air conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: adequate


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 h dark, 12 h light

Study design: in vivo (LLNA)

Vehicle:

other: acetone/olive oil (3:1 v/v)

Concentration:

10%, 50% and 100% (v/v), vehicle served as control.

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
no range finding test was performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Murine Local Lymph Node Assay
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 was determined. The parameter used to characterize the response was 3H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. The increase SI 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice.
The animals were randomly selceted. Identification was ensured by cage number and individual marking (tail).
Body weight determination: The animals were weighed prior to the first application and at the end of the test period.
Clinical observation: Prior to the first aplication and once a day thereafter all animals were observed in order to detect special clinical signs or reaction to treatment.
Form of application: Epicutaneous application is simulating dermal contact with the compound.
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications to the same application site

³H-thymidine injection: Five days after the first topical application of test substance to the ears all mice were injected intravenously (tail vein) with 20 μCi of 3H-methyl ymidine.

Preparation of cell suspension: Approximately 5 hours after 3H-methyl ymidine-injection all animals were sacrificed.
The draining "auricular lymph nodes" was excised, weighed individually, pooled for each animal (2 lymph nodes per animal) and collected in PBS. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamid gauze ( 200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated twice. After the final wash each pellet was resuspended in approx. 3 ml 5% TCA at approx. 4°C overnight for precipitation of macromolecules. Each precipitate was recovered by centrifugation, resuspende in 1 ml 5% TCA and transfered into scintillation vials.
Determination of 3H-methyl-thymidine incorporation of the lymph node cells: The 3H-methyl-thymidine incorporation was measured in a ß-scintillation counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl-thymidine levels were also measured (5% TCA).

Statistics:

EC3 values are determined by linear interpolation between 2 points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of 3. If all measured points ate above or below the stimulation index of 3, no EC3 value can be stated.

Results and discussion

Positive control results:

A concurrent positive control (reliability check) with a known sensitizer was not included into this study. A study using the positive control substance P-Phenylenediamine (CAS 106-50-3) was performed in January 2005 in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.9

Variability:

0.2

Test group / Remarks:

10%

Results 2

Parameter:

SI

Value:

0.9

Variability:

0.4

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

1.5

Variability:

0.5

Test group / Remarks:

100%

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

When applied as 10% and 50% preparation in acetone/olive oil (3:1 v/v), the test substance did not induce relevant changes in the ³H-methyl thymidine incorporation. The 100% test substance preparation caused slight but not concentration related increase in 3H-thymidine incorporation into the lymph node cells, which failed to reach the cut off stimulation index (increase to 3 fold or above of control value =
stimulation index (SI) ≥ 3) and thus lie below the threshold of immunologic relevance. In addition there was no relevant increase in lymph node weights. Thus it is concluded that the test item does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

A skin sensitization test according to OECD 429 was performed. The radioactive Murine Local Lymph Node 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/Ca01aHsd mice each were treated with 10%, 50% and 100% v/v preparations of the test substance in Acetone/Olive Oil (3:1 v/v) or with the vehicle alone. No signs of systemic toxicity were noticed. When applied as 10% and 50% preparation in acetone/olive oil (3:1 v/v), the test substance did not induce relevant changes in the ³H-methyl thymidine incorporation. The 100% test substance preparation caused slight but not concentration related increase in 3H-thymidine incorporation into the lymph node cells, which failed to reach the cut off stimulation index (increase to 3 fold or above of control value = stimulation index (SI) ≥ 3) and thus lie below the threshold of immunologic relevance. In addition there was no relevant increase in lymph node weights. Thus it is concluded that the test item does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.