2,4,6,8-tetramethylcyclotetrasiloxane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-22 to 2010-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An LLNA study was not performed because the test method is not considered to be suitable for substances that contain silicon. Please refer to the attached document for further details.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

An LLNA study was not performed because the test method is not considered to be suitable for substances that contain silicon. Please refer to the attached document for further details.

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6,8-tetramethylcyclotetrasiloxane

EC Number:

219-137-4

EC Name:

2,4,6,8-tetramethylcyclotetrasiloxane

Cas Number:

2370-88-9

Molecular formula:

C4H16O4Si4

IUPAC Name:

2,4,6,8-tetramethyl-1,3,5,7,2,4,6,8-tetroxatetrasilocane

Constituent 2

Reference substance name:

2,4,6,8-Tetramethylcyclotetrasiloxane (supplied as Dow Corning® ZTOMCATSTM CVD Precursor)

IUPAC Name:

2,4,6,8-Tetramethylcyclotetrasiloxane (supplied as Dow Corning® ZTOMCATSTM CVD Precursor)

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

Test System:
Animals: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF
Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM / The Netherlands
Number of Animals for Main Study/Irritation Screening: 30 males / 6 males (Challenge: 20 test animals, 10 control animals; Irritation Screens: 6 animals)
Age at Delivery/Acclimatization Start: 4 to 5 weeks
Body Weight at Acclimatization Start: Test and control animals: 282 to 373 g, Animals used for irritation screens: 281 to 354 g
Identification: By unique cage number and corresponding individual animal number.
Randomization: Randomly selected by hand at time of delivery. No computer randomization.
Acclimatization: Seventeen days under laboratory conditions after health examination. Only animals without any visible signs of illness were used for the study. One to 3 days for the animals used in the irritation screening for induction and challenge. Only animals without any visible signs of illness were used for the study.

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

other: Acetone

Concentration / amount:

Concentration for 3-week induction: 75% (w/w) in acetone
Concentration for challenge: 5% (w/w) in acetone

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: Acetone

Concentration / amount:

Concentration for 3-week induction: 75% (w/w) in acetone
Concentration for challenge: 5% (w/w) in acetone

No. of animals per dose:

1 Irritation Screen for Induction and Challenge I: 3 (nos. 960 - 962)
2 Control Group: 10 (nos. 963 - 972)
3 Test Group: 20 (nos. 973 - 992)
4 Irritation Screen for Induction and Challenge II: 3 (nos. 993 - 995)

Details on study design:

Preparation of Dose Formulation:
The test item and vehicle were placed into a glass beaker on a tared Mettler balance and a weight/weight dilution was prepared. A magnetic stirrer was used to ensure homogeneous distribution of the test item preparation. The preparations were made immediately prior to each dosing. Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer. The test item formulations (in acetone) were applied between 17 and 43 minutes following their preparation. Dose levels are described in terms of material as supplied unless otherwise stated by the Sponsor.

Selection of Test Item Concentration for Main Study:
A number of factors contributed to the selection of the concentrations of test item including irritancy, slope of dose response curve and experience with similar test items. Irritation screenings were conducted to determine the minimal irritating concentration for the induction period and the highest non-irritating concentration for the challenge and re-challenge periods. The results of these screenings are described below.

Epidermal Induction:
For the epidermal induction phase, the concentration selected should produce some irritation but not adversely affect the animals. The selection of the concentration used for the main induction was done after the first irritation screen was performed. During the first irritation screen, mild to moderate skin reactions were observed with the neat test item and the test item formulated at 75%, 50% and 25% in acetone, all applied topically and occlusively. The severity persisted (except in one occasion, at the concentration of 25%) at the 48-hour reading. Based on the first irritation screen results, the test item at 75% in acetone was considered to be the suitable concentration for the 3-week induction in the test group. The test item concentration of 75% in acetone was agreed upon between the Study Director and Sponsor Representative.

Epidermal Challenge:
For the challenge phase, the concentration selected should be the maximum tested non-irritant concentration. As a non-irritant concentration could not be selected in the first irritation screen, a second irritation screen was performed with lower concentrations of 15%, 10%, 5% and 1% in acetone
as well as acetone alone. Discrete/patchy erythema was noted in the second irritation screen on the test sites treated with the test item at 15% and 10% in acetone while no local skin reactions were observed with the test item formulated at 5% and 1% in acetone as well as acetone alone. The non-irritating test item concentration of 5% in acetone required for the challenge was agreed upon between the Study Director and Sponsor Representative.

Rationale:
Dermal administration has historically been used as the route of choice for determining delayed contact hypersensitivity.

Observations:
Viability / Mortality: Daily from delivery of the animals to the termination of the test.
Clinical Signs / Grading of Skin Response: Daily from delivery of the animals to the termination of test. Skin responses were graded during the
irritation screening, induction and challenge periods.
Body Weights: At delivery/acclimatization start, at the end of the irritation screening, at test day 1 (day of treatment) and at the termination of the study.

Necropsy:
No necropsies were performed on the animals of the control and test group sacrificed at termination of their observation period or on the animals of the two irritation screenings sacrificed a few days after their skin assessment was performed. The animals were euthanized by intraperitoneal injection of pentobarbitone at a dose of at least 2.0 mL/kg body weight (equivalent to 324 mg sodium pentobarbitone/kg body weight) and discarded.

Treatment Method:
Patching method: The animal's fur was shaved with a fine clipper blade or equivalent just prior to the exposure. Closed patches were applied to the animals as follows: 0.5 mL of the test item or a freshly prepared test item dilution in a 25 mm Hill Top Chamber (approximately 4.9 cm2 skin exposure). The 25 mm Hill Top Chamber (skin application area of approximately 4.9 cm2) was firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for six hours (± 15 minutes). An identical patching method was used for the irritation screen, induction, challenge and rechallenge phases.

Observation and Scoring:
In order to evaluate the skin reactions following the irritation screenings, challenge and rechallenge, the trunk skin of each animal where the testing patches had been applied was depilated approximately 21 hours after the patches had been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, 4123 Allschwil / Switzerland). The depilation was performed to clean the stratum corneum to facilitate the reading of the possible skin reaction. The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages. The grading method used for the irritation screenings, induction and challenges was identical. It was performed 24 ± 2 hours after removal of the patches for the irritation screening, induction and challenge and repeated 24 ± 2 hours later (48-hour grades) for the irritation screening and
the challenge.

The scoring was performed by visual assessment of erythema and oedema. They were assessed as follows:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
If observed, any other gross lesions not covered by this scoring system were described.

Grading of all animals was done by positioning each animal under true-light (Philips Master TLS HE 28W/840). For evaluation, two parameters were used: the incidence index and the severity index, for both test and control animals. The incidence index is an expression of the number of animals showing a response of grade 1 or greater at the 24- or 48-hour readings out of the total animals in the group. The severity index, designed to describe the intensity of the reaction, is calculated from the total sum of 24- and 48 hour response readings divided by the number of animals exposed.

Statistical Analysis:
Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.

Data Compilation:
The following data were recorded on data sheets and transcribed for compilation and analysis: clinical signs (local/systemic), mortality/viability and skin reactions. The following data were recorded on-line: body weights. The RCC Tox Computer System (RCC-Tox-Lims) has been validated with respect to data collection, storage and retrievability.

Challenge controls:

10 naive control animals were challenged at the same test item concentration like the 10 test group animals.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE (Harlan Laboratories Study C67807)

Results and discussion

Positive control results:

General:
The purpose of this skin sensitizing study was to confirm the allergenic potential of ALPHAHEXYLCINNAMALDEHYDE when administered topically to albino Dunkin Hartley guinea pigs. For this purpose the “Buehler Test” modified by Ritz, H.L. and Buehler, E.V. (1980) was used. Twenty male animals of the test group were treated topically with ALPHAHEXYLCINNAMALDEHYDE at 50% in PEG 300 once a week for a 3-week induction phase. Two weeks after the final induction application the animals were challenged with the same test item concentration of 5% in PEG 300 as used for induction. The ten animals of the control group were not treated during the induction. They were treated once at challenge with ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300.

Results:
None of the control animals were observed with skin reactions after the challenge treatment with the highest tested non-irritating concentration of ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300.
Nine and six of twenty test animals were observed with discrete/patchy erythema at the 24- and 48-hour readings, respectively, after the challenge treatment with the highest tested non-irritating concentration of ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300. No skin effect was observed in the control group.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

5% (w/w) in acetone

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5% (w/w) in acetone

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

5% (w/w) in acetone

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5% (w/w) in acetone

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

ALPHA-HEXYLCINNAMALDEHYDE at 5%

No. with + reactions:

9

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

ALPHA-HEXYLCINNAMALDEHYDE at 5%

No. with + reactions:

6

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

RESULTS AND DISCUSSION

Observations

Viability / Mortality / Macroscopic Findings

There were no deaths during the course of the study and no necropsies were performed.

Clinical Signs

No signs of systemic toxicity were observed in any of the animals.

Body Weights

(See attached Summary Tables)

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Skin Reactions in the Induction

(See attached Individual Tables)

No skin effect (grade 0) was observed in the control group after treatment with acetone alone during the three weeks of induction.

Discrete/patchy erythema (grade 1) was observed in all (100%) animals in the first and third induction weeks and in 17 out of 20 (85%) animals in the second induction week after treatment with the test item at 75% in acetone.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitisation study, conducted according to OECD 406 and in compliance with GLP, concluded that 2,4,6,8-tetramethylcyclotetrasiloxane is not sensitising to skin.

Executive summary:

General:

The purpose of this skin sensitizing study was to assess the ability of the test item, 2,4,6,8 -tetramethylcyclotetrasiloxane, to induce delayed contact hypersensitivity when applied topically to albino guinea pigs. A modified Buehler method (Ritz and Buehler, 1980) was used. The study was conducted according to OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization”.

Twenty male animals of the test group were treated topically with 2,4,6,8 -tetramethylcyclotetrasiloxane at 75% in acetone once per week for a 3-week induction phase. Ten animals in the control group were treated in the same way as the test animals, but with the vehicle (acetone) only. Two weeks after the final induction application the control and test animals were challenged with the test item at 5% in acetone and acetone alone.

Challenge:

There was no skin reaction in the control and test group after the challenge procedure performed with 2,4,6,8 -tetramethylcyclotetrasiloxane at 5% in acetone. The acetone, itself, did not show any local skin reactions and was, therefore, considered to be suitable for this type of study.