3-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]- 2-hydroxypropyl methacrylate and 1-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-3-hydroxypropan-2-yl methacrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15th -30th September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

10-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-11-yl 2-methylprop-2-enoate; 11-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-10-yl 2-methylprop-2-enoate

EC Number:

700-334-3

Molecular formula:

C17H38O6Si3

IUPAC Name:

10-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-11-yl 2-methylprop-2-enoate; 11-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-10-yl 2-methylprop-2-enoate

Test material form:

other: liquid

Details on test material:

Sponsor's identification: SiMAA2
Description : Clear colourless liquid
Batch number : 003068
Date received : 25 March 2008
Storage conditions: Approximately 4°C in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Halan UK limited, Bicester, Oxon, UK.
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended soild-floor polypropylene cages furnished with softwood woodflakes. Animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (e.g. ad libitum): Free assess to food supplied by Harlan Teklad.
- Water (e.g. ad libitum):Free assess to mains tap water
- Acclimation period: 5 days
-Other: nullparious and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 Deg C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light (06:00-18:00) and 12 hours darkness (18:00 - 06:00).

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Groups of four mice per dose level were treated with the undiluted test material or the test material at concentrations of 50% or 25% in acetone/olive oil 4:1.

No. of animals per dose:

4 animals per dose level.

Details on study design:

Preparation of Test Material:
For the purpose of the study, the test material was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration


PROCEDURE
In the main test, one animal treated with the undiluted test material showed signs of systemic toxicity, pre-dose on Day 2, and was therefore killed for humane reasons due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence. The absence of this animal for the remainder of the study was considered not to affect the purpose or integrity of the study.


PRELIMINARY SCREENING TEST:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test material 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. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.


MAIN TEST:
Test material administration:
Groups of four mice were treated with the undiluted test material or the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for up to three consecutive days (Days 1, 2, 3). The test material 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 material (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, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Observations:
Clinical Observations: Animals were observed twice daily on Day 1, pre-dose on Day 2 and the surviving animals post-dose on Day 2, twice daily on Day 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.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination), or at death.

TERMINAL PROCEDURES:
Termination: Approximately five hours following the administration of 3HTdR the surviving mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the 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 labelled with the project 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 ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty 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).

Interpretation of Results:
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 material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".

Positive control substance(s):

other: alpha-Hexylcinnamaldehyde, Tech, 85%

Results and discussion

Positive control results:

One group of five animals was treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

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

Concentration % v/v in acetone/olive oil 4:1: 15
Stimulation index: 10.91
Result: Positive.

alpha-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.89

Test group / Remarks:

25%

Remarks on result:

other: negative

Results 2

Parameter:

SI

Value:

4.72

Test group / Remarks:

50%

Remarks on result:

other: positive

Results 3

Parameter:

SI

Value:

6.69

Test group / Remarks:

100% (undlluted)

Remarks on result:

other: positive

Results 4

Parameter:

other: Disintergrations per minute (dpm)

Value:

6 343.22

Test group / Remarks:

Vehicle

Results 5

Parameter:

other: Disintergrations per minute (dpm)

Value:

12 019.06

Test group / Remarks:

25%

Results 6

Parameter:

other: Disintergrations per minute (dpm)

Value:

29 928.53

Test group / Remarks:

50%

Results 7

Parameter:

other: Disintergrations per minute (dpm)

Value:

31 846.38

Test group / Remarks:

100% (undiluted)

Any other information on results incl. tables

Preliminary Screening Test:

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were100%, 50% and 25% v/v in acetone/olive oil 4:1.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in table below:

A stimulation index of greater than 3 was recorded for the two concentrations of the test material (100% and 50% v/v in acetone/olive oil 4:1).

A stimulation index of less than 3 was recorded for the lowest concentration of the test material (25% v/v in acetone/olive oil 4:1).

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

Concentration (%v/v) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	6343.22	792.90	na	na
25	12019.06	1502.38	1.89	Negative
50	29928.53	3741.07	4.72	Positive
100	31846.38	5307.73*	6.69	Positive

 

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

* =        Dpm/node obtained by dividing the Dpm value by 6 (due to the death of one animal on Day 2)

na =      Not applicable

Clinical Observations and Mortality Data

One animal, treated with the undiluted test material, was killed for humane reasonsdue to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted were hunched posture, lethargy, ptosis and splayed gait. A 2g bodyweight loss was also noted. No other signs of systemic toxicity were noted in the surviving test or control animals during the test.

Bodyweight:

Bodyweight changes of the surviving test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test.

Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

- OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted)

- Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% v/v, 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 material or the test material as asolutioninacetone/olive oil 4:1at concentrations of 50% or 25% v/v. A further group of four animals was treated withacetone/olive oil 4:1alone.

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) in acetone/olive oil 4:1	Stimulation Index	Result
25	1.89	Negative
50	4.72	Positive
100	6.69	Positive

Conclusion. 

The test material was considered to be a sensitiser under the conditions of the test.