1-Butanamine, N-[3-(trimethoxysilyl)propyl]-, reaction products with (trimethoxysilyl)-N-[(trimethoxysilyl)propyl]-1-propanamine and 1,6-diisocyanatohexane homopolymer

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

iGloss Crosslinker (ZQ54-2211)

IUPAC Name:

iGloss Crosslinker (ZQ54-2211)

Details on test material:

- Name of test material (as cited in study report): iGloss Crosslinker (ZQ54-2211)
- Test-substance No.: 11/0715-1
- Lot/batch No.: 294222
- Purity: Mn 1200 Da; Mw 1800 Da; PDI 1.5; Relative RI-area of compounds with M 250-500 Da: 1.7% and with M 250-1000 Da: 29.1%; Determined with Gel Permeation Chromatography
- Physical state, appearance: Yellowish, clear liquid
- Homogeneity: The test substance appeared to be homogeneous
- Storage conditions: At room temperature, protected from light and moisture
- Expiration date: February 01, 2013

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst, Netherlands
- Age at study initiation: 11-12 weeks (pre-test 1), 8-9 weeks (pre-test 2), 9-10 weeks (pre-test 3), 9-10 weeks (main test)
- Weight at study initiation: 18.3-21.8 g
- Housing: Group housing in Makrolon Type II (pre-tests)/ III (main study), with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) with granulated soft wood bedding (Rettenmaier & Sohne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 28 - 65 (acclimation period); 30 - 65 (main study)
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 2.5, 5, 10% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: At a concentration of 50% the test item could be suspended in vehicle. At a test item concentration of 25% and below, the test item could be dissolved in the vehicle.
- Irritation: In the first pre-test, animals showed erythema of the ear skin (animal treated with 50%: from day 3 to 6 Score 1, animal treated with 100%: from day 2 to 4 Score 1 and on days 5 and 6 Score 2). At both tested concentrations, an increase in ear weight was observed that exceeded the threshold value of 25% for excessive local skin irritation. Additionally, at the high dose, an increase in ear thickness was observed that exceeded the threshold value of 25% for excessive local skin irritation. Furthermore, in the group treated with 100% of the test item, strong hair loss was observed behind the ears (day 5) and on day 6, raw ear skin and excoriated epidermis was observed. Additionally, at 50%, the skin was peeling off (day 6).
In the second pre-test concentrations of 10 and 25% (w/w) were used. On day 3 and 4, an erythema of the ear skin was observed in the animal treated with 25% test item concentration (Score 1). At 25% test item concentration, an increase in ear thickness was observed that exceeded the threshold value of 25% for excessive local skin irritation. At 10% test item concentration, 21.6% increase in ear thickness was still observed.
A third pre-test was performed with test item concentrations of 5 and 2.5% (w/w). On day 3 and 6, an erythema of the ear skin was observed in the animal treated with 5% (Score 1). The ear weight and ear thickness measurements did not show a distinct increase.
- Lymph node proliferation response: Not determined.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Preparation
The test item was placed into an appropriate container on a tared balance and acetone:olive oil (4+1 v/v) was quantitatively added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil (4+1 v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. Two further groups of mice (one vehicle control group and one positive control group) were treated with an equivalent volume of the relevant vehicle alone or with the positive control item at 25% (w/v).

Administration of 3H-methyl thymidine
3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytics, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL).
Five days after the first topical application (day 6) 250 pL of phosphate-buffered saline (PBS) containing 19.7 NCi of 3HTdR (equivalent to 78.8 NCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium (Release®, WDT, 30827 Garbsen, Germany). The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of Ultima Gold scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed. The level of 3HTdR incorporation was then measured on a ß-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 8.3.3 was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (Casy Cell Counter, Innovatis). The values obtained were taken down manually.

Determination of Ear Weights
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Interpretation of Raw Data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response (see Ref. 8) and the cutoff-value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1. However, these cutoff-values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.

Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: At least once daily from experimental start to necropsy.
- Body weights: In the pre-tests: prior to the first application and prior to sacrifice (with exception of animal 1 in the second pre-test). In the main experiment: prior to the first application and prior to treatment with 3HTdR.
- Ear thickness: In the pre-tests prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany).
- Ear weights: In the pre-tests and main experiment after sacrifice; biopsy punches were taken from each ear.
- Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
- Lymph node cell count: The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
- Clinical signs (local / systemic): Recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
The EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb s test were used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/w) Stimulation index Result 0 1.00 - 2.5 2.81 positive 5 5.96 positive 10 10.36 positive

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Concentration (% w/v) Disintegrations per minute 0 479.3 2.5 1348.9 5 2855.3 10 4965.3

Any other information on results incl. tables

Viability/Mortality

No deaths occurred during the study period.

 

Clinical Signs

No signs of systemic toxicity were observed during the study period. On day 2, an erythema of the ear skin was observed in all animals treated with 10% test item concentration (Score 1).

 

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

 

Lymph Node Weights and Cell Counts

The measured lymph node weights and -cell counts of all animals treated were recorded after sacrifice.

A statistically significant increase in mean lymph node weight and lymph node cell count was observed in all test item treated groups in comparison to the vehicle control group (p<0.05). A clear dose response was observed in both parameters.

For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count were exceed in the mid and high dose group (index of 2.1 and 2.8, respectively).

A statistically significant increase in lymph node weight and -cell count (p<0.05) was observed in the positive control group in comparison to the acetone:olive oil (4+1 v/v) vehicle control group. Also, the above mentioned cutoff-value for a positive response of the lymph node cell count index was exceeded in this group (index: 2.5).

 

Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was observed in the low and the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cutoff-value of 1.1 was reported for a positive response of the ear weight index regarding ear skin irritation. The indices determined for the low and high dose group exceeded this threshold (indices of 1.2).

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU