Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 05 Dezember 2012 to 11 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Aminopropyl Vinyl Ether
- Physical state: Liquid, colourless, clear
- Analytical purity: 99.6 corrected area-%
- Lot/batch No.: C520/070/2012/2
- Expiration date of the lot/batch: June 27, 2016
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Ambient (RT), avoid temperatures >25°C
- Homogeneity: The test substance was homogeneous by visual inspection.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Animal strain: Mouse / CBA/CaOlaHsd
- Mean weight at study initiation: ca. 19.6 - 22.3 g
- Housing: Single housing; Makrolon cage, type II
- Bedding: H15005-29 Ssniff Spezialitaeten GmbH
- Diet: STANRAB (P) SQC; SDS Special Diets Services, Altrip, Germany
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30- 70
- 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:
5%, 2% and 1% (w/w)
No. of animals per dose:
5
Details on study design:
In order to study a possible skin sensitizing potential of Aminopropyl Vinyl Ether, three groups each of five female mice were treated with different
concentrations of the test item by topical application at the dorsum of each ear once daily each on three consecutive days. A control group of five
mice was treated with the vehicle only. The sensitivity and reliability of the experimental technique employed was assessed by an additional positive
control group of five mice. The positive control experiment was performed with 25% (w/w) α-hexyl cinnamaldehyde dissolved in acetone: olive oil (4:1 v/v) (compound listed in OECD 442B Guideline). Four days after the first topical application, the mice were intraperitoneally injected with BrdU. Approximately 24 hours after intraperitoneally injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and
immediately weighed using an analytical balance. Furthermore, both ears of mice were punched at the apical area using a biopsy punch and the punches were immediately weighed pooled per animal using an analytical balance. Afterwards, single cell suspensions of lymph node cells were prepared
from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. The proliferative
capacity of pooled lymph node cells was determined by the incorporation of BrdU measured in a photometer.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 1, 2, and 5% (w/w) in AOO. The application volume 25 μL/ear/day was spread over the entire dorsal surface (Æ~ 8 mm) of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was
treated with 25 % α-hexyl cinnamaldehyde dissolved in acetone: olive oil (4:1 v/v).
BrdU was purchased from Roche Diagnostics GmbH. For injection it was dissolved in DPBS (10 mg/ml) before administration and stored in a refrigeratoruntil use. Four days after the first topical application (day 5) 0.5 ml of BrdU solution (5mg/per mouse/injection) were intraperitoneally injected.
Approximately 24 hours after treatment with BrdU the mice were sacrificed and 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 a 70 μmnylon mesh. The lymph node cells were resuspended in 6 ml DPBS. After cell count with a cell counter (Casy Cell Counter, Innovatis), cell
suspensions of 100 000 cells / ml were adjusted. The incorporation of BrdU into lymph node cells was determined using a commercial cell
proliferation assay kit as follows (Roche Applied Science. Mannheim): 100 μl of the lymph node cell suspension (100 000 cells /ml) were added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the lymph node cells with FixDenat (included inCell Proliferation Elisa Kit), anti BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution TMB (included inCell Proliferation Elisa Kit) was then added and allowed to produce chromogen. After stopping the substrate reaction the
absorbance was measured at 450 nm with a reference wavelength of 690 nm (Absorbance-Reader SUNRISE, Tecan).
Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance (XS 204, Mettler Toledo, serial number 112 748 2217). Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 9.3.3 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 (XS 204, Mettler Toledo).
Interpretation of Raw Data
The proliferative response of the lymph node cells is expressed as the mean SI. The SI is derived by dividing the mean BrdU labeling index/mouse within each test substance group as well as for the positive control group by the mean BrdU labeling index for the vehicle group.
A test item is regarded as a sensitizer 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 BrdU at least 1.6-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.Furthermore, according to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation.

Observations:
Mortality / Viability: At least once daily from experimental start to necropsy. Clinical signs (local / systemic) Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.
Body weights: Pre-test and main experiment: prior to the first application and prior to sacrifice.
Ear thickness: In the pre-test prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer (S0247 Kroeplin).
Ear weights: In the pre-test 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.








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 BrdU values.
For the calculations excel (Version 2010) was applied.
The EC1.6 value was calculated according to the equation
EC1.6 = (a-c) [(1.6-d)/(b-d)] + c
where EC1.6 is the estimated concentration of the test item required to produce a 1.6-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 1.6 on the local lymph node assay dose response plot. For the calculations excel (Version 2010) was applied.
A statistical analysis was conducted on the BrdU 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 Statistica (Version 10) was used. A Mann-Whitney-U test for non-parametric comparison was applied as statistical test. Statistical significance was set at the five per
cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: In this study Stimulation Indices (S.I.) of 1.7, 1.1 and 2.4 were determined with the test item at concentrations of 1, 2 and 5% (w/w) in AOO, respectively. Due to the fact that no clear dose response was observed an EC1.6 could not be calculated.

Any other information on results incl. tables

Stimulation Index. BrdU labeling Index

Test item concentration
% (w/w)

Group Calculation

Mean BrdU

labeling indexa)

S.I.

Vehicle (AOO)

0.085

1.0

1 % (test substance)

0.143

1.7

2 % (test substance)

0.090

1.1

5 % (test substance)

0.204

2.4

25%Positive control (HCA)

0.288

3.4

a) Mean BrdU labeling index was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals per group)

S.I. = Stimulation Index (values of the test item groups related to the mean value of the control group)

 

 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test item Aminopropyl Vinyl Ether was found to be a skin sensitizer under the test conditions of this study.
Executive summary:

In this study the test item Aminopropyl Vinyl Ether was assessed for its skin sensitizing potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone : olive oil (AOO, 4:1, v/v).

The local lymph node assay is recommended by international test guidelines (e.g. OECD) as an animal test for predicting skin sensitization in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitizers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is the Stimulation Index (S.I.). BrdU labeling is used to measure cell proliferation.

For this purpose a local lymph node assay was performed using test item concentrations of 1, 2 and 5% (w/w; weight per weight). Doses based on previously performed three pretests in the same animal strain in which 18 mice in total were used. In the pre-tests the highest concentration that could be technically used was 100% (w/w). Signs of systemic toxicity were not observed in the pre-tests.

At the tested concentrations of 10, 25, 50 and 100% the animals showed signs of local irritation as confirmed by the ear weight measurements. At least one of two animals in these dose groups showed increased ear weights > 25%. In addition, animals in dose

group 25, 50 and 100% showed ear swellings > 25%. Moreover hardening of the ears, moderate ear swelling and very slight erythema was observed in dose group 50 and 100 %. Hardening of the ears was also noted in dose group 25%. Furthermore very slight erythema and incrustations were observed in dose group 10 and 25%. Animals in dose group 2.5 and 5% didn`t show increased ear weights > 25% or increased ear thickness. In both groups scaling was noted. After consulting the sponsor, the following dose levels were selected for the main study: 5%, 2% and 1% (w/w) in AOO. In the main study the animals did not show any relevant signs of systemic toxicity. Very slight erythema and incrustations were observed in the high dose (5%) group. According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group.

A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentration results in a 1.6-fold or greater increase in incorporation of BrdU compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 1.6 is referred to as the EC1.6 value.

In this study Stimulation Indices (S.I.) of 1.7, 1.1 and 2.4 were determined with the test item at concentrations of 1, 2 and 5% (w/w) in AOO, respectively. Due to the fact that no clear dose response was observed an EC1.6 could not be calculated. A statistically significant and biologically relevant increase in BrdU labeling was observed for the low (1%) and high (5%) dose group. This was also observed for these dose groups regarding lymph node weights and lymph node cell count, thus corroborating the sensitizing potential. In the 2% group only lymph node cell count reached statistically significance. Furthermore, the cut-off-value for a positive response regarding the lymph node cell countindex of 1.55 reported for BALB/c mice was exceeded in the low and high dose group (index of 1.6 (1%) and 1.9 (5%)). As expected, a statistical and biological relevant increase in BrdU labelling, lymph node weight and lymph node cell count was determined in the positive control. The test item Aminopropyl Vinyl Ether was thus a skin sensitizer under the test conditions of this study.