2-Propenoic acid, 2-methyl-, tris(1-methylethyl)silyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July to 3 August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent study conducted by a GLP certified laboratory in accordance with a suitable study guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Tripropan-2-ylsilyl 2-methylprop-2-enoate

EC Number:

700-182-8

Cas Number:

134652-60-1

Molecular formula:

C13 H26 O2 Si

IUPAC Name:

Tripropan-2-ylsilyl 2-methylprop-2-enoate

Details on test material:

- Name of test material (as cited in study report): TIS-M
- Lot/batch No.: 8211
- Expiration date of the lot/batch: 12 June 2010
- Storage condition of test material: Under nitrogen at room temperature, protected from light.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France.
- Age at study initiation: approx. 12 weeks old.
- Weight at study initiation: Body weight variation within +/- 20% of the sex mean.
- Housing: Animals were individually housed in Macrolon cages (MI type, height: 12.5cm). Sterilised sawdust was used as bedding material (Litalobo, S.P.P.S, Argenteui, France) and paper was supplied as cage-enrichment (Enviro-dri, Wm. Lillico and Son (Wonham Mill Ltd.), Surrey, United Kingdom). The paper was removed on day 1 prior to dosing and was supplied again after scoring of the ears on day 3.
- Diet: SM R/M-Z from SSNIF® Spezialdiäten GmbH, Soest, Germany; ad libitum
- Water: Tap water; ad libitum.
- Acclimation period: At least 5 days before treatment. Accomodation was the same but the Macrolon cages were MIII type, height 18cm.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-23.5°C
- Humidity (%): 40-70% (actual range 41-76%). Cleaning procedures in the room might have caused temporary fluctuations above the optimal maximumof 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
- Air changes (per hr): approx. 15
- Photoperiod: 12 hours light, 12 hours dark.

IN-LIFE DATES: From: 15 July To: 3 August 2009

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0% (vehicle only), 25%, 50% and 100% concentrations.

No. of animals per dose:

5

Details on study design:

Preliminary irritation study

A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2) at theh ighest concentration.

Two test substance concentrations were tested; a 100% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (undiluted for liquids).

The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Main study

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (25 uL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.
The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Excision of the nodes - Day 6

All animals:
Each animal was injected via the tail vein with 0.25 ml_ of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 uCi of 3H-methyl thymidine (GE Health care, Buckinghamshire, UK).

After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). 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 areawere recorded. The nodes were pooled for each animal in approximately 3 ml_ PBS.

Tissue processing for radioactivity - Day 6

A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 urn). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4°C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) 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 of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). 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).

Observations

Mortality /Viability Twice daily.
Toxicity At least once daily.
Body weights On Days 1 (pre-treatment) and 6.
Irritation On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The Stimulation Index (SI) is the ratio of the DPM/group compared to the DPM/vehicle group. If the results indicate a SI ≥ 3, the substance may be regarded as a skin sensitiser. Consideration was also given to the EC3 value (the estimated test substance concentration that will give SI = 3. The SI values calculated for the substance concentrations 25. 50 and 100% were 18.7, 40.8 and 35.7 respectively. These results inidcate that the test substance could elict a SI ≥ 3. The response of the 100% group did not follow the expected dose-response relationship which is more often seen in these kind of studies. The response might be less due to differences in skin penetration (no vehicle present) or viscosity or due to underlying systematic toxicity. No reliable EC3 value could be calculated. As the SI values clearly exceeded 3, TIS-M was determined to be a skin sensitiser.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM values are presented for each animal and each dose group below.

Any other information on results incl. tables

Table 1: Skin reactions after epidermal exposure and body weights (Preliminary Study):

Animal number	Test Substance (% w/w)	Day 1	Day 3
		Body Weight (g)	Skin reactions dorsal surface ear				Body weight (g)
			Left		Right
			erythema	oedema	erythema	oedema
1	50	26	1	0	1	0	24
2	100	23	1	0	1	0	20

- Vehicle: Methyl ethyl ketone

Table 2: Skin reactions, body weights and relative size auricular lymph nodes (Main Study):

Group	Test Substance (% w/w)	Animal Number	Day 1	Day 3				Day 6
			Body Weight (g)	Skin reactions dorsal surface ear				Body Weight (g)	Size nodes
				Left		Right
				erythema	oedema	erythema	oedema		left	right
1	0% (vehicle)	1	25	0	0	0	0	24	n	n
		2	24	0	0	0	0	23	n	n
		3	22	0	0	0	0	23	n	n
		4	24	0	0	0	0	24	n	n
		5	23	0	0	0	0	23	n	n
2	25%	6	26	1	0	1	0	24	+	n
		7	23	1	0	1	0	23	n	+
		8	25	1	0	1	0	25	n	n
		9	24	1	0	1	0	24	+	+
		10	22	1	0	1	0	22	n	n
3	50%	11	23	1	0	1	0	22	++	++
		12	25	1	0	1	0	23	++	++
		13	23	1	0	1	0	23	+	+
		14	25	1	0	1	0	23	++	++
		15	22	1	0	1	0	21	+	+
4	100%	16	23	1	0	1	0	22	+	+
		17	25	1	0	1	0	24	++	++
		18	22	1	0	1	0	21	+	+
		19	25	1	0	1	0	24	+	+
		20	26	1	0	1	0	25	++	++

- Vehicle: Methyl ethyl ketone

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

Table 3: Radioactivity measurements (individual animals):

Group	Test substance (% w/w)	Animal number	DPM/animal
1	0% (vehicle)	1	158
		2	211
		3	136
		4	147
		5	254
2	25%	6	3720
		7	2710
		8	4638
		9	5786
		10	117
3	50%	11	5484
		12	9622
		13	5787
		14	7816
		15	8292
4	100%	16	4496
		17	7225
		18	29*
		19	9698
		20	4479

- Vehicle: Methyl ethyl ketone

- * = value rejected (activity too low to represent an acceptable level of activity for this study).

Table 4: Disintegrations Per Minute (DPM) and Stimulation Index (SI):

Group	Test substance (% w/w)	Mean DPM± SEM	SI±SEM
2	25%	3394±964	18.7±5.8
3	50%	7400±780	40.8±6.6
4	100%	6475±1253	35.7±8.2
1	0% (vehicle)	181±22	1.0 ± 0.2

- Vehicle: Methyl ethyl ketone

Preliminary irritation study: (Table 1):

The concentration selected for the main study was the highest concentration: 100% concentration.

Main study: Skin reactions/irritation: (Table 2):

The slight irritation of the ears as shown by all experimental animals was considered not to have a toxicologically significant effect on the activity of the nodes.

Main study: Macroscopy of the auricular lymph nodes and surrounding area: (Table 2):

The majority of the auricular lymph nodes were condsidered to be enlarged. No macroscopic abnormalities of the surrounding area were noted.

Main study: Body weights: (Table 2):

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not to be toxicologically significant.

Main study: Radioactivity measurements (Tables 3 and 4):

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 3394, 7400 and 6475 respectively . The mean DPM/animal value for the vehicle control group was 181.

No mortality occured and no symptoms of systematic toxicity were observed in the animals of the main study.

Reliability check results: (Table and graphs in attached background material):

The SI values calculated for the susbtance concentrations 5, 10 and 25% were 2.0, 2.2 and 4.2 respectively. An EC3 value of 16.0% was calculated using linear interpolation.The calculated EC3 value was found to be in the acceptable range of 2 -20%. The results of the 6 monthly HCA reliability checks in recent years were 13.1, 15.6, 14.1, 13.8 and 13.9%. Based on the results, it was concluded that the Local Lymph Node Assay as performed by NOTOX is an appropriate model for testing for contact hypersensitivity.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The registered substance was determined to be a skin sensitiser, as the SI values were all above 3.

Executive summary:

A skin sensitivity study of the substance was carried out according to EU Method B.42 (Local Lymph Node Assay) using CBA mice.

The registered substance was applied to the dorsal surface of both ears and after 6 days the substance was determined to be a skin sensitiser.