Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-24 to 2008-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Hungarian Supplier: WOBE Kereskedelmi Kft., H-1164, Budapest, Garmada u. 10.

- Age at study initiation: Young adult, 9-10 weeks old, age-matched within one week
- Housing: Individual caging
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H , ad libitum
- Water (e.g. ad libitum): tap water from municipal supply, ad libitum
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 8-12 air exchange/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25; 50; 100 % (w/v) (25µl)
No. of animals per dose:
4
Details on study design:
Dose selection:
The selection of treatment concentrations based on results of a preliminary irritation toxicity test. This test was performed in groups of 2 CBNCa mice with test item concentrations of 100 % (undiluted test item) and 50 % (% w/v) in the selected vehicle (AOO). This preliminary test was conducted in a similar experimental manner to the exposure phase of the main study.
No mortality or signs of systemic toxicity were observed during the preliminary test. No treatment related effect on body weights was observed. No cutaneous reaction was observed in any of the treated groups. The observations recorded in this preliminary test suggest that the formulations, the application of the material and the local effects on the animal are acceptable for a valid LLNA.

In the main assay sixteen female CBA/Ca mice were allocated to four groups of four animals each:
- Three groups received the appropriate formulation of SIKA Hardener LI at concentrations of 100 %, 50 % or 25 %,
- the negative control group received the vehicle (AOO).
Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

Clinical observations:
During the study (from Day 1 to Day 6) each animal was observed at least once daily for any clinical signs, including local irritation and systemic toxicity. Individual records were maintained.
Measurement of body weight:
Individual body weights were recorded on Day 1 (beginning of the test) and at Day 6 (prior to ³HTdR injection) with a precision of +/- 0.1 g.
Evaluation of the results:
DPM was measured for each group. The results were expressed as DPN (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN of a treated group divided by DPN of the negative control group) for each treatment group was also calculated.
Interpretation of results:
The test item is regarded as a sensitiser if both of the following criteria are fulfilled:
- Exposure to at least one concentration of the test item result in an incorporation (tritiated methyl thymidine) of at least 3-fold or greater than recorded in control mice
- 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.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
NA

Results and discussion

Positive control results:
The positive control substance α-Hexylcinnamaldehyde (HCA) was examined at a concentration of 25 % in the relevant vehicle. A significant lymphoproliferative response (SI ≥ 3) was noted for HCA with stimulation index value of 11.3 in accordance with the historical data, demonstrating the test reliability.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A significant lymphoproliferative response (SI ≥ 3) was noted for SIKA Hardener LI at all of the applied concentrations. The stimulation index values were 6.2, 4.8 and 4.1 at concentrations of 100 % (undiluted test item), 50 % and 25 %, respectively. The stimulation index values were compatible with the conventional biological dose-response. An EC3 value was extrapolated from the data according to Annex IV of the Updated ICCVAM-Recommended Protocol. A value of 8.4 % was extrapolated from the raw data.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Group DPN (measured DPNgroup values – average DPN background values) obtained for the negative control group, SIKA Hardener LI 100%, 50%, 25% groups were 1876.3, 11638.0, 9023.2, 7638.9 respectively. Or expressed as DPN/node with the following results 234.5, 1454.8, 1127.9, 954.9, obtained in the negative control group, SIKA Hardener LI 100%, 50%, 25% groups respectively.

Any other information on results incl. tables

clinical signs and mortality:

No mortality or signs of systemic toxicity were observed during the study. No significant effects were observed on animal body weights. No irritation or other cutaneous effect was observed in any of the groups treated.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of the present assay, SIKA Hardener LI as supplied and in a suitable vehicle, was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of SIKA Hardener LI following dermal exposure in the Local Lymph Node Assay. The assay was performed with CBNCa Ola Hsd female mice according to the OECD guideline 429 and the Commission Directive 2004/73/EC, method B.42. Vehicle compatibility was verified in a preliminary test with Acetone: Olive oil 4:1 mixture (AOO), in which the test item was completely miscible. A preliminary irritation toxicity test was performed with the test item at concentrations of 100 % (the undiluted test item) and 50 % (%w/v) in the selected vehicle.

In the main assay sixteen female CBN Ca mice were allocated to four groups of four animals each: Three groups received the appropriate formulation of SIKA Hardener LI at concentrations of 100 %, 50 % or 25 %, and the negative control group received the vehicle (AOO). Each concentration was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on days 4, 5 and 6. On day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine. Obtained values were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No treatment related effect on the body weights was observed. No signs of irritation or other cutaneous effect were observed in any of the treated groups.

A significant lymphoproliferative response (SI > 3) was noted for SIKA Hardener LI at all of the applied concentrations. The stimulation index values (SI) were 6.2, 4.8 and 4.1 at concentrations of 100 %, 50 % and 25 %, respectively. The stimulation index values obtained were compatible with the conventional biological dose-response. An EC3 value was extrapolated from the data according to Annex IV of the Updated ICCVAM-Recommended Protocol. A value of 8.4 % was extrapolated from the raw data. The result of the latest reliability check was used to demonstrate the appropriate performance of the assay in accordance with the OECD guideline 429. The result with the positive control substance a-Hexylcinnamaldehyde (HCA) confirmed the validity of the LLNA in this laboratory.

It was concluded that under the conditions of the present test, SIKA Hardener LI has a sensitisation potential (sensitiser) in the Local Lymph Node Assay.