Dimethoxymethylsilane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan - 08 Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

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:

Dimethoxymethylsilane

EC Number:

240-914-9

EC Name:

Dimethoxymethylsilane

Cas Number:

16881-77-9

Molecular formula:

C3H10O2Si

IUPAC Name:

Dimethoxy(methyl)silane

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-Quality
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 19.5 to 23.9 g
- Housing: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap-water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no; before the initiation of dosing, a health inspection was performed, and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 37 - 46 (the value that was outside the targeted range (40 - 70%) occurred on one day only and was without a noticeable effect on the clinical condition of the animals or on the outcome of the study)
- Air changes (per hr): ≥ 10
- 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% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: the test item was soluble in the vehicle at 50% (w/w)
- Irritation: no irritation was observed
- Systemic toxicity: no signs of systemic toxicity were noted
- Ear thickness measurements: see Table 2 (any other information on results, incl. tables)
- Erythema scores: see Table 1 (any other information on results, incl. tables)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine
- Criteria used to consider a positive response: if the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer

TREATMENT PREPARATION AND ADMINISTRATION:
Induction (Days 1, 2 and 3): the dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The formulations were stirred with a magnetic stirrer until dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Lymph Nodes (Day 6): each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized according to laboratories Standard Operating Procedures. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity (Day 6): following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements (Day 7): precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

IN-LIFE PROCEDURES, OBSERVATIONS, AND MEASUREMENTS
- Mortality/Moribundity Checks: animals were observed for general health/mortality and moribundity twice daily.
- Clinical Observations: post-dose observations were performed once daily on Days 1 - 6 (on Days 1 - 3 at least 3 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.
- Body Weights: animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy).
- Irritation: erythema and eschar formation observations were performed once daily on Days 1 - 6 (on Days 1 - 3 within 1 hour after dosing).

Positive control substance(s):

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

Results and discussion

Positive control results:

A study (performed in November 2021; Charles River Laboratories Den Bosch BV) was performed to assess the sensitivity of the CBA/J mice at the testing facility to a known sensitizer. The positive control substance alpha hexylcinnamaldehyde (25% (v/v) in acetone:olive oil (4:1)) was considered to be a sensitizer under the conditions of the test (SI 5.7; EC3 12.2%).
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 12.8, 9.0, 10.9, 8.0 and 13.5.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.9

Variability:

± 0.3

Test group / Remarks:

100%

Results 2

Parameter:

SI

Value:

0.5

Variability:

± 0.1

Test group / Remarks:

50%

Results 3

Parameter:

SI

Value:

0.7

Variability:

± 0.1

Test group / Remarks:

25%

Results 4

Parameter:

SI

Value:

1

Variability:

± 0.2

Test group / Remarks:

vehicle control

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 259, 193 and 331 DPM, respectively. The mean DPM/animal value for the vehicle control group was 362 DPM.

DETAILS ON STIMULATION INDEX CALCULATION: the SI values calculated for the test item concentrations 25, 50 and 100% were 0.7, 0.5 and 0.9, respectively.

EC3 CALCULATION: the EC3 value could not be calculated, since all SI's are below the threshold value of 3.

CLINICAL OBSERVATIONS: no mortality occurred and no clinical signs of systemic toxicity were observed in the animals.

BODY WEIGHTS: body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SIGNS OF TOXICITY: no irritation was observed in any of the animals.

MACROSCOPIC EXAMINATION OF LYMPH NODES AND SURROUNDING AREA: all auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.
Two animals dosed at 100% showed an abnormality of the auricular lymph nodes (adhesion to the vena jugularis and/or small nodules on the surface). No macroscopic abnormalities of the surrounding area were noted for the other animals.

Any other information on results incl. tables

Table 1: Pre-Screen Test - Body Weights and Skin Reactions

TI (%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
		bw (g)	Erythema		Erythema		Erythema		Erythema		Erythema		Erythema		bw (g)
			left	right	left	right	left	right	left	right	left	right	left	right
50	81	27.0	0	0	0	0	0	0	0	0	0	0	0	0	24.1
	82	23.6	0	0	0	0	0	0	0	0	0	0	0	0	21.9
100	83	23.3	0	0	0	0	0	0	0	0	0	0	0	0	23.5
	84	25.5	0	0	0	0	0	0	0	0	0	0	0	0	25.4

TI = test item (% w/w)

bw = body weight (grams)

Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear); 0 = No erythema

 

Table 2: Pre-Screen Test - Ear Thickness Measurements

TI (%)	Animal	Day 1		Day 3				Day 6
		Left	Right	Left		Right		Left		Right
		(mm)	(mm)	(mm)	%	(mm)	%	(mm)	%	(mm)	%
50	81	0.200	0.205	0.220	10	0.225	10	0.230	15	0.245	20
	82	0.205	0.200	0.220	7	0.230	15	0.230	12	0.235	18
100	83	0.210	0.205	0.225	7	0.230	12	0.255	21	0.230	12
	84	0.210	0.210	0.230	10	0.235	12	0.230	10	0.235	12

Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters

TI = test item (% w/w)

% = Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for use in the main study.

 

Table 3: Main Study - Body Weights and Skin Reactions

Group	TI(%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			bw (g)	Erythema^#		Erythema		Erythema		Erythema		Erythema		Erythema		bw (g)
				left	right	left	right	left	right	left	right	left	right	left	right
1	0	1	22.3	0	0	0	0	0	0	0	0	0	0	0	0	22.3
		2	21.7	0	0	0	0	0	0	0	0	0	0	0	0	21.9
		3	20.5	0	0	0	0	0	0	0	0	0	0	0	0	21.5
		4	20.6	0	0	0	0	0	0	0	0	0	0	0	0	20.5
		5	19.5	0	0	0	0	0	0	0	0	0	0	0	0	20.6
2	25	6	22.7	0	0	0	0	0	0	0	0	0	0	0	0	24.1
		7	23.9	0	0	0	0	0	0	0	0	0	0	0	0	24.2
		8	20.1	0	0	0	0	0	0	0	0	0	0	0	0	20.2
		9	22.7	0	0	0	0	0	0	0	0	0	0	0	0	23.1
		10	21.9	0	0	0	0	0	0	0	0	0	0	0	0	21.3
3	50	11	22.9	0	0	0	0	0	0	0	0	0	0	0	0	23.4
		12	20.6	0	0	0	0	0	0	0	0	0	0	0	0	21.3
		13	20.5	0	0	0	0	0	0	0	0	0	0	0	0	20.9
		14	23.0	0	0	0	0	0	0	0	0	0	0	0	0	23.5
		15	20.8	0	0	0	0	0	0	0	0	0	0	0	0	22.0
4	100	16	20.8	0	0	0	0	0	0	0	0	0	0	0	0	20.7
		17	22.3	0	0	0	0	0	0	0	0	0	0	0	0	21.9
		18	22.6	0	0	0	0	0	0	0	0	0	0	0	0	22.8
		19	20.0	0	0	0	0	0	0	0	0	0	0	0	0	20.8
		20	21.6	0	0	0	0	0	0	0	0	0	0	0	0	22.2

TI = test item (% w/w).

bw = body weight (grams).

^# Grading erythema and eschar formation (Left = dorsal surface of left ear; right = dorsal surface of right ear); 0 = No erythema

 

Table 4: Main Study - Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

Group	TI (%)	Animal	Size Nodes		DPM  / Animal	Mean DPM ± SEM			Mean SI ± SEM
			left	right
1	0	1	n	n	289	362	±	65	1.0	±	0.2
		2	n	n	357
		3	n	n	309
		4	n	n	243
		5	n	n	610
2	25	6	n	n	172	259	±	26	0.7	±	0.1
		7	n	n	299
		8	n	n	325
		9	n	n	244
		10	n	n	254
3	50	11	n	n	187	193	±	50	0.5	±	0.1
		12	n	n	142
		13	n	n	215
		14	n	n	60
		15	n	n	361
4	100	16	n	n	197	331	±	91	0.9	±	0.3
		17	n^#	n^#	229
		18	n	n	437
		19	n	n^#$	154
		20	n	n	636

TI = test item (% w/w).

Size Nodes: Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal)

DPM = Disintegrations per minute

SEM = Standard Error of the Mean

SI = Stimulation index

^#   Small nodules on the surface

^$   Adhesion to vena jugularis

 

Table 5: Positive Control Reliability Check (Nov 2021)

Group	% HCA	Mean DPM ± SEM	SI ± SEM
1	0% (AcOO)	281 ± 19	1.0 ± 0.1
2	5%	410 ± 54	1.5 ± 0.2
3	10%	795 ± 45	2.8 ± 0.1
4	25%	1593 ± 162	5.7 ± 0.5

AcOO = Acetone/Olive oil (4:1 v/v)

HCA = Alpha- Hexylcinnamaldehyde

DPM = Disintegrations per minute

SEM = Standard Error of the Mean

SI = Stimulation index

 

 

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

The study was conducted according to OECD 429 and in compliance with GLP. Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%, dimethoxy(methyl)silane was considered not to be a skin sensitizer.