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Diss Factsheets

Administrative data

Description of key information

LLNA (OECD 429): not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 - 15 Feb 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
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: 20.2 to 24.2 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 (mean)
- Humidity (%): 37 - 46 (the value that was outside the targeted range (40 - 70%) occurred for one day 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
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-screen test: 50 and 100%
Main test: 25, 50 and 100%
No. of animals per dose:
Pre-screen test: 2
Main test: 5
Details on study design:
PRE-SCREEN TESTS:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
- Compound solubility: no data
- Irritation: no irritation was observed
- Systemic toxicity: no signs of systemic toxicity were noted
- Ear thickness measurements: yes, prior to dosing on Days 1 and 3, and on Day 6 using a digital thickness gauge (see Table 2 in Any other information on results, incl. tables)
- Erythema scores: see Table 1 in Any other information on results, incl. tables

MAIN STUDY
- 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) 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 200 g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) 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)
Positive control results:
A reliability check was carried out in Nov 2021 to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. The SI values calculated for the alpha-hexylcinnamaldehyde concentrations 5, 10 and 25% were 1.5, 2.8 and 5.7, respectively. An EC3 value of 12.2% was calculated using linear interpolation. 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.
Parameter:
SI
Value:
1
Variability:
± 0.1
Test group / Remarks:
vehicle control
Parameter:
SI
Value:
0.8
Variability:
± 0.1
Test group / Remarks:
25%
Parameter:
SI
Value:
1.1
Variability:
± 0.3
Test group / Remarks:
50%
Parameter:
SI
Value:
2
Variability:
± 0.6
Test group / Remarks:
100%
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 133, 178 and 335 DPM, respectively. The mean DPM/animal value for the vehicle control group was 165 DPM.

DETAILS ON STIMULATION INDEX CALCULATION: The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean (see Table 3 in Any other information on results, incl. tables).

EC3 CALCULATION: The EC3 value could not be calculated, since all S.I.´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. The moderate body weight loss noted for one animal (No. 12) was considered not test item-related since it concerned only one animal and no dose-related incidence was apparent.

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.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

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

21.7

0

0

0

0

0

0

0

0

0

0

0

0

22.2

82

22.3

0

0

0

0

0

0

0

0

0

0

0

0

21.4

100

83

21.2

0

0

0

0

0

0

0

0

0

0

0

0

20.3

84

20.5

0

0

0

0

0

0

0

0

0

0

0

0

20.5

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

0.220

0.225

0.230

5

0.235

4

0.225

2

0.225

0

82

0.215

0.220

0.225

5

0.230

5

0.230

7

0.220

0

100

83

0.205

0.210

0.215

5

0.220

5

0.225

10

0.220

5

84

0.215

0.210

0.225

5

0.225

7

0.230

7

0.230

10

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

24.0

0

0

0

0

0

0

0

0

0

0

0

0

23.0

2

22.9

0

0

0

0

0

0

0

0

0

0

0

0

23.3

3

21.4

0

0

0

0

0

0

0

0

0

0

0

0

21.3

4

22.8

0

0

0

0

0

0

0

0

0

0

0

0

22.7

5

21.7

0

0

0

0

0

0

0

0

0

0

0

0

21.2

2

25

6

23.3

0

0

0

0

0

0

0

0

0

0

0

0

23.3

7

24.2

0

0

0

0

0

0

0

0

0

0

0

0

24.1

8

23.1

0

0

0

0

0

0

0

0

0

0

0

0

22.5

9

23.0

0

0

0

0

0

0

0

0

0

0

0

0

24.0

10

23.7

0

0

0

0

0

0

0

0

0

0

0

0

24.2

3

50

11

22.1

0

0

0

0

0

0

0

0

0

0

0

0

21.7

12

22.3

0

0

0

0

0

0

0

0

0

0

0

0

20.2

13

20.7

0

0

0

0

0

0

0

0

0

0

0

0

21.6

14

21.7

0

0

0

0

0

0

0

0

0

0

0

0

21.6

15

20.2

0

0

0

0

0

0

0

0

0

0

0

0

21.4

4

100

16

22.1

0

0

0

0

0

0

0

0

0

0

0

0

21.5

17

23.1

0

0

0

0

0

0

0

0

0

0

0

0

22.6

18

21.2

0

0

0

0

0

0

0

0

0

0

0

0

20.2

19

21.7

0

0

0

0

0

0

0

0

0

0

0

0

21.3

20

22.1

0

0

0

0

0

0

0

0

0

0

0

0

21.5

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

138

165± 23

1.0 ± 0.1

2

n

n

221

3

n

n

212

4

n

n

154

5

n

n

102

2

25

6

n

n

91

133±19

0.8± 0.1

7

n

n

105

8

n

n

125

9

n

n

144

10

n

n

201

3

50

11

n

n

74

178± 55

1.1± 0.3

12

n

n

111

13

n

n

190

14

n

n

129

15

n

n

386

4

100

16

n

n

162

335± 102

2.0± 0.6

17

n

n

165

18

n

n

239

19

n

n

408

20

n

n

702

TI = test item (% w/w).

DPM= Disintegrations per minute.

SEM = Standard Error of the Mean.

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

 

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

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
In a LLNA according to OECD 429 and in compliance with GLP, there was no indication that the test item elicited a SI ≥ 3 when tested up to 100%. Thus, the test item was considered not to be a skin sensitizer.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitization potential of dimethoxy(dimethyl)silane was examined in a LLNA according to OECD 429 and in compliance with GLP. Test item concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v) (AcOO)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 133, 178 and 335 DPM, respectively. The mean DPM/animal value for the vehicle control group was 165 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 0.8, 1.1 and 2.0, respectively.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Since there was no indication that the test item elicited a SI 3 when tested up to 100%, dimethoxy(dimethyl)silane was considered not to be a skin sensitizer.

 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification of the registered substance.