Hexyltrimethoxysilane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 2018 to 07 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Hexyltrimethoxysilane

EC Number:

221-331-9

EC Name:

Hexyltrimethoxysilane

Cas Number:

3069-19-0

Molecular formula:

C9H22O3Si

IUPAC Name:

hexyltrimethoxysilane

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, 5800 AN Venray, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9–10 weeks
- Housing: animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet available ad libitum
- Water: tap water, sulphur acidified to a pH value of approx. 2.8 available ad libitum
- Acclimation period: 13 days

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

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25%, 50% (each diluted in AOO 4:1, v/v) and 100% (undiluted test item)

No. of animals per dose:

5 mice / test group; 5 mice / control group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item was found to be 100%. The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO.
- Irritation: The mice were observed daily for any clinical signs of local irritation at the application site. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%. Neither signs of irritation nor excessive irritation at any application site could be detected in any animal.
- Systemic toxicity: The mice were observed daily for any clinical signs of systemic toxicity at the application site. Body weights were recorded pre-test and prior to termination. Cage-side observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge). No signs of systemic toxicity at any application site could be detected in any animal. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the duration of the prescreen test.
- Ear thickness measurements: Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6.
- Erythema scores: Both ears were observed for erythema and scored on a scale of 0 (no erythema) to 4 (severe erythema to eschar formation preventing grading of erytema). All animals were valued at erythema score 0.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION: Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application. The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR). Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1. Five days after the first topical application all mice were dosed with 3H-methyl thymidine by intravenous injection (tail vein) of 3H-methyl thymidine, diluted with phosphate buffered saline (PBS). Approximately 5 hours after the injection, all mice were sacrificed. The auricular lymph nodes were 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 gauze. 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. After the final wash each pellet was resuspended at approx. 4° C for approximately 18 hours for precipitation. Each precipitate was washed again, resuspended, and transferred into vials to be stored at room temperature overnight. The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured. Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: 1% phenylenediamine

Statistics:

None reported

Results and discussion

Positive control results:

The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

2.5

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

3.2

Test group / Remarks:

100%

Results 4

Key result

Parameter:

EC3

Remarks:

%

Value:

85.71

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The means of the lymph node weights per group showed relevant difference in the 50% and 100% test groups compared to the negative control. The mean weight of the lymph nodes were 2.9 mg at 25%, 4.5 mg at 50%, and 5.3 mg at 100%. The mean weight of the lymph nodes for the control group was 3.0 mg.

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation indices were 0.8 at 25%, 2.5 at 50%, and 3.2 at 100%.

EC3 CALCULATION
The EC3 value was calculated to be at a test item concentration of 85.71 %, when using the SI values at the concentrations of 50% and 100%.

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS
All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study.

Applicant's summary and conclusion

Interpretation of results:

other: Category 1B based on CLP criteria (EU criteria according to Regulation (EC) No. 1272/2008).

Conclusions:

In a local lymph node assay conducted according to OECD 429 and to GLP (reliability score 1), the test material is considered to be a skin sensitiser.