Diethoxy(dimethyl)silane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Diethoxy(dimethyl)silane

EC Number:

201-127-6

EC Name:

Diethoxy(dimethyl)silane

Cas Number:

78-62-6

Molecular formula:

C6H16O2Si

IUPAC Name:

diethoxydimethylsilane

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 5222A20170420
- Purity test date: 20 Apr 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator.
- Stability under test conditions: The stability of the test substance under storage conditions over the study period is guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: Good solubility of the test substance in the vehicle MEK was achieved. The stability of the test substance in the vehicle was determined indirectly by analysis of the homogeneity / concentration control analysis.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was produced on a weight per weight (w/w) basis in the vehicle MEK (methyl ethyl ketone) shortly before application. The test- substance preperations were solubilized by a short stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

Mouse / CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH, C/O Postfach 553, NL-5800 AN Venray.
- Females (if applicable) nulliparous and non-pregnant: not specified.
- Age at study initiation: 8 weeks (pretest, 10% and 50% test subtance prepration), 9 weeks (pretest, undiluted test substance), 8 weeks (main test).
- Weight at study initiation: 18.7 g – 20.9 g (pretest), 17.3 g – 21.7 g (main test).
- Housing: Polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: at least 5 days before the first test substance application.
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C.
- Humidity (%): 45 – 65%.
- Photoperiod (hrs dark / hrs light): 12 hours light from 06:00 h to 18:00 h; 12 hours darkness from 18:00 h to 06:00 h.
- IN-LIFE DATES: From: 30 Jan 2018 To: 05 Feb 2018.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0, 25%, 50% in MEK, undiluted test substance
The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local irritation as determined in the pretest.

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The highest test substance concentration used in the pretest was the undiluted test substance (100%).
- Irritation: At the tested concentrations, the animals did not show any signs of local irritation as confirmed by the ear weight (compared to historical vehicle values) and ear thickness measurements.
- Systemic toxicity: No signs of systemic toxicity were observed in the pretest.
- Ear thickness measurements: Yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response:
A test item is regarded as sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H-thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. lymph node cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL test substance or test substance preparations were applied to the dorsal surfaces of both ears.

Positive control substance(s):

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

Statistics:

WILCOXON - Test: 3H-thymidine incorporation, cell count, lymph node weight and ear weight

Results and discussion

Positive control results:

Periodic positive controls, most current values from Jan 2018:
Alpha-Hexylcinnamaldehyde (techn. 85%) tested in concentrations of 1%, 5%, 15% in the vehicle MEK yielded Stimulation Indeces 3H-thymidine incorporation of 1.59, 3.17, 7.48 and Stimulation Indeces Cell counts of 1.27, 1.66 and 2.83, respectively.
The EC 3.0 and EC 1.5 values were 4.6 and 3.3, respectively.

In vivo (LLNA)

Results

Parameter:

EC3

Value:

> 100

Remarks on result:

other: no increase above the cutoff Stimulation Index of 3 up to the highest tested concentration of 100%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: Statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, of the auricular lymph node cell counts and of the lymph node weights. The applied test-substance concentrations did not cause any increase in ear weights demonstrating the absence of relevant ear skin irritation.

DETAILS ON STIMULATION INDEX CALCULATION: The mean stimulation indices (expressed as multiples of the vehicle control or of the untreated control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized in Table 1.

EC3 CALCULATION: The applied test-substance concentrations did not induce a biologically relevant (no increase above the cutoff Stimulation Index of 3). Therefore, the EC3 is > 100%.

CLINICAL OBSERVATIONS: No signs of systemic toxicity were noticed in all animals during general observation.

BODY WEIGHTS: No influence on the body weights was observed during the study.

Any other information on results incl. tables

Table 1: Stimulation Indices

Test Group	Treatment	^3H-thymidine incorporation Stimulation Index^1	Cell Count Stimulation Index^1	Lymph Node Weight Stimulation Index^1	Ear Weight Stimulation Index^1
0	untreated	1.00	1.00	1.00	1.00
1	vehicle MEK	1.00	1.00	1.00	1.00
2	25% in MEK	1.63 #	1.07	1.11	0.96
3	50% in MEK	2.41 ##	1.38 ##	1.35 ##	1.02
4	undiluted	2.7 ##	1.52 ##	1.61 ##	1.06

¹test group 2 -3 vs. test group 1 (vehicle control) // test group 4 vs. test group 0 (untreated control)

The statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.05, ## for p≤ 0.01)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay.

Executive summary:

The skin sensitizing potential of the test material 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 cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 25% and 50% (w/w) preparations of the test substance in MEK, with the undiluted test substance, or with the vehicle alone or not treated at all.

The study used 3 test groups and 2 control groups. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation or of the undiluted test substance applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone.

Three days after the last application, 20 µCi³H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated measuring³H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation.

The applied test-substance concentrations did not induce a biologically relevant (no increase above the cutoff Stimulation Index of 3), but statistically significant and concentration-dependent increase of ³H-thymidine incorporation into the cells from the auricular lymph nodes.

When applied undiluted the test substance induced a concentration-dependent, statistically significant response just above the cut-off for biological relevance (increase to 1.5-fold or above of control value = stimulation index (SI)≥1.5) in the auricular lymph node cell counts, as well as a biologically relevant, concentration dependent and statistically significant increase in lymph node weights.

The applied test-substance concentration did not cause any increase in ear weights demonstrating the absence of relevant ear skin irritation.

 

Thus, it is concluded that the test material does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay underthe test conditions chosen.