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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:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
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

Constituent 1
Chemical structure
Reference substance name:
1,3-Propanediol, 2-methyl-, reaction products with ethenyltrimethoxysilane
EC Number:
700-957-0
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 β-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)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The mean stimulation index values of the test substance were 4.75, 2.57 and 1.71 at treatment concentrations of 100%, 50% and 25%, respectively. A significant increase in the stimulation index compared to controls was observed after treatment with 100% of the test substance (see Table 2 under "Any other information on results incl. table"). Based on these results, an EC3 value of 59.9% (w/v) was calculated (see Table 3 under "Any other information on results incl. table").
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
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 value (average of the 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 per group of 520.8, 784.6 and 1450.2, respectively. A statistically significant increase in DPM/lymph node (1450.2) compared to control values (DPM/node = 305.3) after treatment with 100% of the test substance and a clear dose-response relationship were observed (see Table 1 under "Any other information on results incl. table").

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:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: Skin Sens 1B, H317
DSD: Xi, R43
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.