1,3-Propanediol, 2-methyl-, reaction products with ethenyltrimethoxysilane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 19 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-Propanediol, 2-methyl-, reaction products with ethenyltrimethoxysilane

EC Number:

700-957-0

Cas Number:

1141852-17-6

Molecular formula:

UVCB

IUPAC Name:

1,3-Propanediol, 2-methyl-, reaction products with ethenyltrimethoxysilane

Constituent 2

Reference substance name:

Reaction products of trimethoxy(vinyl)silane and 2-methylpropane-1,3-diol (2:5-6)

IUPAC Name:

Reaction products of trimethoxy(vinyl)silane and 2-methylpropane-1,3-diol (2:5-6)

Details on test material:

- Name of test material (as cited in study report): Y-15866
- Analytical purity: 72%
- Lot/batch No.: 3710-10
- Expiration date of the lot/batch: 2013-07-16
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., AD Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 21.2 g (animals of the range-finding test) and 21.8 g (animals of the main study)
- Housing: individually in Makrolon Type II cages with wire mesh top (EHRET GmbH, Emmendingen, Germany)
- Diet: pelleted standard diet (Harlan Laboratories B.V., AD Horst, The Netherlands), ad libitum
- Water: tap water (Gemeindewerke, Rossdorf, Germany), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25 and 50% (w/v), and 100%

No. of animals per dose:

Range-finding test: 1 (in test groups)
Main study: 5 (controls), 5 (in test groups)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the compound solubility was determined according to the recommendations given in OECD guideline 429. The highest test substance concentration which can be technically used was 100% (undiluted). Further dilutions were performed in dimethylformamide (DMF).
- Irritation: to determine the highest non-irritant dose that does at the same time not induce signs of systemic toxicity, two mice were treated once daily on three consecutive days with concentrations of 50% (w/v) and 100% by topical (epidermal) application to the dorsal surface of each ear. Clinical signs were recorded within 1 h and 24 ± 4 h after each application as well as on Day 7. In addition, the initial and terminal body weights of each animal were determined. The animals did not show any signs of irritation or systemic toxicity after test substance application at concentrations of 50 and 100%. Based on these results, concentrations selected for the main study were 25, 50 and 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine (³HTdR) incorporation determined by ß-scintillation
- Criteria used to consider a positive response: the test substance is considered a skin sensitiser in the LLNA if the following criteria were met:
(1) the exposure to at least one concentration of the test substance resulted in an incorporation of ³HTdR at least 3-fold greater than in controls (as indicated by the stimulation index) and
(2) the data were compatible with a conventional dose response, although allowance had to be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: each animal was treated by topical application of 25 µL of the different test substance concentrations and the respective vehicle (DMF as vehicle control) on the entire dorsal surface of each ear. The application was repeated on Days 2 and 3. On Day 6, each animal was treated with 250 µL of 80.9 µCi/mL ³HTdR (corresponds to 20.2 µCi ³HTdR per mouse) in phosphate buffered saline (PBS) by intravenous injection via the tail vein. After five hours, mice were euthanised by intraperitoneal injection of sodium pentobarbital and the draining auricular lymph nodes were rapidly excised. The 2 nodes of each animal were pooled and single cell suspensions (SCS) in PBS were prepared by gentle mechanical disaggregation of each pooled lymph node cells (LNCs) through a stainless steel gauze (200 µm mesh size). LNCs were pelleted by centrifugation, and washed twice with PBS. After removal of the washing solution, pellets were resuspended in 5% trichloroacetic acid and incubated at 4 °C for approximately 18 h for precipitation of macromolecules.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations of body weights and DPM values were calculated. Statistical analysis of the mean DPM values was performed between treated and control groups to assess a dose-response relationship.

Results and discussion

Positive control results:

A significant increase in the stimulation indices (SI = 3) was noted for the positive control substance hexyl cinnamic aldehyde in acetone:olive oil (4+1) at concentrations of 10% (SI = 3.4) and 25% (SI = 6.1), respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Value:

59.9

Results 2

Parameter:

SI

Value:

1.71

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

2.57

Test group / Remarks:

50%

Results 4

Parameter:

SI

Value:

4.75

Test group / Remarks:

100%

Cellular proliferation data / Observations:

Two lymph nodes of each animal were pooled and DPM values were measured from the pooled lymph node cell suspensions. The measured DPM values per animal were corrected by subtracting the background DPM values (average of two measured DPM values of 5% (w/v) trichloroacetic acid solutions). Treatment with 25, 50 and 100% test substance in DMF resulted in DPM/lymph node of 520.8, 784.6 and 1450.2, respectively.

Any other information on results incl. tables

No systems of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Table 1. Calculation of individual DPM and SI values

Test substance concentr.			DPM values measured	DPM-BG per animal (2 lymph nodes)*	SI
%	Animal No.	Group No.
-	-	-	13 (BG I)	-	-
-	-	-	12 (BG II)	-	-
-	1	1	530	518	-
-	2	1	778	766	-
-	3	1	511	499	-
-	4	1	483	471	-
-	5	1	813	801	-
25 (w/v)	6	2	811	799	1.3
25 (w/v)	7	2	837	825	1.4
25 (w/v)	8	2	1306	1294	2.1
25 (w/v)	9	2	396	384	0.6
25 (w/v)	10	2	1920	1908	3.1
50 (w/v)	11	3	1288	1276	2.1
50 (w/v)	12	3	2031	2019	3.3
50 (w/v)	13	3	2217	2205	3.6
50 (w/v)	14	3	1748	1736	2.8
50 (w/v)	15	3	624	612	1
100	16	4	5466	5454	8.9
100	17	4	2288	2276	3.7
100	18	4	1565	1553	2.5
100	19	4	2982	2970	4.9
100	20	4	2263	2251	3.7

BG = background (1 mL 5% trichloroacetic acid)

1 = control group; 2-4: test groups

SI = stimulation index relative to the mean of the control group (group 1)

* values corrected for mean background value (BG I and BG II)

Table 2. Calculation of mean DPM and SI values

Test substance concentration	DPM per lymph node#	SI
Vehicle	305.3	1.00
25%	520.8	1.71
50%	784.6	2.57
100%	1450.2*	4.75

# DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a group by the number of lymph nodes taken from that group.

*Statistically significant compared to control (p = 0.005)

Table 3. Calculation of the EC3 value

Test group	Test substance concentration	SI
3	50% (a)	2.57 (b)
4	100% (c)	4.75 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 59.9 %

EC3 = estimated concentration for a SI of 3

a, b, c, d = co-ordinates for the 2 pairs of data lying immediately above and below the SI value of 3 on the LLNA dose response plot

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of an LLNA, performed according to OECD/EC guidelines, Y-15866 is concluded to be a skin sensitiser. As the LLNA is not considered a suitable test for silicone materials, the result is disregarded and further testing to address this endpoint has been initiated.

Executive summary:

The skin sensitising potential of Y-15866 was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD guideline 429 and in compliance with GLP. Based on a preliminary test in two female CBA/CaOlaHsd mice, the undiluted test substance (100%) and dilutions of 25 and 50% (w/v) of the test substance in dimethylformamide (DMF) were selected as treatment concentrations for the main study. In this experiment, 5 female CBA/CaOlaHsd mice per test group were treated with the undiluted or diluted test substance or vehicle alone, respectively. The test substance formulations or the vehicle were applied on the external surface of each ear (25 µL/ear) for three consecutive days. Five days after the first topical application, the cell proliferation of pooled lymph nodes from individual animals was measured by incorporation of ³H-methyl thymidine and expressed as the amount of radioactive disintegration per minute (DPM). The mean DPM/lymph node for each test group was 520.8, 784.6 and 1450.2 at concentrations of 25, 50 and 100% of the test substance, respectively. Treatment with the undiluted test substance (100% concentration) resulted in a statistically significant increase in DPM/lymph node (1450.2) compared to control values (DPM/node = 305.3) and showed a clear dose-response relationship. Based on these results, stimulation indices of 4.75, 2.57 and 1.71 were calculated for the treatment concentrations of 100%, 50% and 25%, respectively. The estimated concentration for a stimulation index of 3 (EC3) was 59.9%. No local or systemic toxicity and no effects on body weights were observed. The historical positive control hexyl cinnamic aldehyde confirmed the sensitivity and reliability of the experimental technique (SI = 3). Under the above mentioned conditions, the test substance was found to be a sensitiser in the LLNA.