9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 26 September 2016 and 18 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

220July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC 440/2008

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:

9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

EC Number:

289-390-3

EC Name:

9-[2-(2-methoxyethoxy)ethoxy]-9-[3-(oxiranylmethoxy)propyl]-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Cas Number:

88127-84-8

Molecular formula:

C21H44O11Si

IUPAC Name:

9-[2-(2-methoxyethoxy)ethoxy]-9-{3-[(oxiran-2-yl)methoxy]propyl}-2,5,8,10,13,16-hexaoxa-9-silaheptadecane

Test material form:

liquid

Remarks:

clear colorless

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd strain

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 26 September 2016 To: 18 October 2016

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Preliminary screening test: 25µL undiluted test item.

Main Test: undiluted test item or 50% or 25% w/v in dimethyl formamaide

No. of animals per dose:

Prelilminary test = 1
Main test = 4 per dose

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Table 1 (below). Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test

Test Item Administration
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures

Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes.
The pellet was re suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 ”C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by  scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".
The results were also evaluated according to the Globally Harmonized Classification System.

Positive control substance(s):

not specified

Results and discussion

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

> 1.12 - < 2.27

Remarks on result:

other: Non-sensitizer

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in dimethyl formamide	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	6564.67	820.58	na	na
25	7384.78	923.10	1.12	Negative
50	8123.43	1015.43	1.24	Negative
100	14902.20	1862.78	2.27	Negative

dpm =  Disintegrations per minute

^a =       Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

^b =       Stimulation Index of 3.0 or greater indicates a positive result

na  =    Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitizer under the conditions of the test.
The test item does not meet the criteria for classification according to the Globally Harmonized Classification System

Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.  Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in dimethyl formamide at concentrations of 50% or 25% v/v.  A further group of four animals was treated with dimethyl formamide alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) indimethyl formamide	Stimulation Index	Result
25	1.12	Negative
50	1.24	Negative
100	2.27	Negative