Methyl-tris acetonoximo-silane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-10-04 to 2004-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 24 April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

EC Number:

460-110-3

EC Name:

A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

IUPAC Name:

A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

Specific details on test material used for the study:

- Name: "WASOX-MMAC2"
- Batch No.: 1000024854
- Purity: The test substance is a mixture of mainly 3 components:
MMAC2 (range: 45-80% w/w), accurately 55.0% (GC-% w/w),
MM2AC (range 2-30% w/w), accurately 11.7% (GC-% w/w) and
MAC3 (range 5-30% w/w),accurately 24.0% (GC-% w/w) plus by-products and impurities.
The 3 components act uniformly as hardeners for silicone sealing masses. They polymerise, triggered bv hydrolysis.
- CAS No. (main component): 72122-57-7
- Solubility in water: Poorly soluble. A polymeric, insoluble in water, is formed by hydrolysis. Rapid onset of hydrolysis.
- Solubility in other solvents: Easily soluble in toluene and methylcyclohexane
- Appearance: Solid and liquid parts at room temperature.Yellowish brownish colour.
- Melting point: 30 - 35 °C.
- Conditions of storage: Storage under a nitrogen atmosphere, as the substance reacts with water, even from air humidity. Ambient temperature (theoretically up to approx. 40 °C possible), protected against light (handling without protection against light is acceptable).
- Handling precautions: Exothermic reaction with water or humidity from the air.
- Date of expiry: December 2005

In vivo test system

Test animals

Species:

mouse

Strain:

other:

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelman GmbH, 33176 Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: About 8 weeks at the first administration
- Weight at study initiation: 17.0-19.9 g
- Housing: Single caging in Makrolon cages type II
- Diet (e.g. ad libitum): Yes, Altromin maintenance diet for rats and mice, item No.1324forte
- Water (e.g. ad libitum): Yes, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Average of 21.8
- Humidity (%): Average of 48.2
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 50 and 100%

No. of animals per dose:

5

Details on study design:

MAIN STUDY
The test substance was administered in 3 concentrations to the dorsal surfaces of the ears of the animals of the test substance groups. In a manner identical to that of animals in the treatment groups animals of one negative control group and one positive control group were treated with AOO and HCA respectively. Each animal was treated for 3 consecutive days. 3 days after the last administration the proliferation of the Iymphocytes of the draining Iymph nodes was measured by the determination of the amounts of incorporated 3HTdR.

Administration of the test substance:
Route of Administration: epicutaneous administration to the dorsal surface of each ear
Administration volume: 25 µL

Incorporation of 3H-methyl thymidine in vivo:
5 days after the first topical administration, each animal received 20 µCi 3HTdR by slow intravenous administration. The injection solution containing nominal 80 µCi/mL 3HTdR was prepared by the dilution of 1.152 mL 3HTdR (amersham pharmacia biotech, Kat. Nr.:TRA 310, batch 315, specific activity 2.0 Ci/mmol, radioactive concentration 1 mCi/mL, radiochemical purity 98.6%, thymine content 0.2%) with 13.248 mL PBS. Several minutes prior to 3HTdR administration the animals were kept restrained in Plexiglas-tubes and the tail veins were visualised by placing the tails in warm water. Then 250 µL of the injection solution were intravenously administered to each animal.

Preparation of single cell suspensions and determination of incorporated 3H-methyl thymidine:
Approximately 5 hours after 3HTdR injection all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised. The Iymph nodes of each group were pooled in PBS. A single cell suspension (SCS) of Iymphnode cells (LNC) nwas prepared by gentle mechanical disaggregation of the pooled Iymph nodes through a 70 µm cell strainer. The SCSs were then transferred into centrifuge tubes and LNC were pelleted by centrifugation (4 °C, max. 200 g, 10 min). Afterwards supernatants were removed by aspiration. Then the LNC were resuspended and washed twice with PBS. After the final washing the supernatants were removed leaving just a small volume < 0.5 mL) and
macromolecules were precipitated by incubation with 5 % trichloroacetic acid (TCA) at 4 °C overnight. Each precipitate was pelleted by centrifugation (4 °C, max. 200 g, 10min) and resuspended in 1 mL TCA. This suspension was transferred into scintillation vials containing 10 mL scintillation cocktail (Packard Bioscience: Ultima Gold, Bestellnr. 6013329) and 3HTdR incorporation was determined with a ß-scintillation counter.

INVESTIGATIONS
General observations: All animals were observed at least once daily for behavioural changes or signs of toxicity
Body masses: The body mass of each animal was recorded on Days 1 and 6
Skin reactions: The application sites were visually checked for local irritations once a day from Days 1-6.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Calculation of the stimulation indices (Sls):
Results are presented as test/control ratio (Stimulation index), calculated as dpm test group/dpm negative control group.
(dpm = disintegrations per minute, corrected by the subtraction of the background)

Results and discussion

Positive control results:

Application of 25% HCA in AOO resulted in an SI of 10.2. This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

25%

Remarks on result:

other: no indication of sensitisation

Results 2

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

50%

Remarks on result:

other: no indication of sensitisation

Results 3

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

100%

Remarks on result:

other: no indication of sensitisation

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION : The SIs of the test substance groups were between 0.7 and 1. For detailed results please refer to table 1 in box "Any other information on results incl. tables".

SKIN REACTIONS: No local skin irritations were observed at the application sites of all animals of all test substance groups and both control groups throughout the whole study.

MORTALITY: No mortality occurred

GENERAL OBSERVATIONS: One animal of the high dose group had reduced motoric activities on Day 6. In all other animals no abnormal behaviour or clinical signs were detected during the experiment.

BODY MASS: Body masses and body mass gains of all animals were in the range to be expected from animals of the same strain, sex and age.

Any other information on results incl. tables

Table 1: DPM results and calculated SIs

Test group	DPM	SI
Negative control	4197	1
Low dose	4217	1.0
Mid dose	3536	0.8
High dose	2919	0.7
Positive control	42767	10.2

Rationale for selection of the concentrations:

In a range finding study the test substance as it is (100%) was administered to two animals. In this study the animals were treated in the same manner as in a main study with 25 µL test substance on the dorsum of each ear on three consecutive days. None of the animals showed overt systemic toxicity, excessive local skin irritation or an increase in ear thickness in the range finding study. Therefore, 100% was chosen as highest test substance concentration and ear thickness measurement was not performed in the main study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance "WASOX-MMAC2" is not sensitizing under the conditions of this LLNA study (OECD 429).

Executive summary:

In a dermal sensitization study conducted according to OECD 429 with "WASOX-MMAC2" suspended in AOO (4:1 v/v acetone/olive oil), young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 25% (v/v), 50% (v/v) and 100% (undiluted) in a local lymph node assay (LLNA). There was no mortality nor clinical observations nor effects on body weights observed. None of the tested concentrations of the test substance reached the stimulation index (SI) threshold of 3. Therefore, no EC3 value could be determined. Application of the positive control substance hexyl cinnamic aldehyde (25%) resulted in a SI of 10.2. This result proves the sensitivity of the strain of animals used and the reliability of the experimental technique. In this study, the test substance is not a dermal sensitizer.