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Description of key information

SIKA Hardener MH was tested for skin sensitising properties in a local lymph node assay LLNA. Based on the results obtained from the test, the substance was considered as a skin sensitiser in Cat. 1.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2011-05-31 to 2011-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. H-1103, Budapest, Cserkesz u. 90
- Age at study initiation: Young adult mice
- Weight at study initiation: The weight variation in animals involved in the study will not exceed ± 20 % of the mean weight.
- Housing: grouped caging (4 animals/cage); type II. polypropylene/polycarbonate
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water, as for human consumption, from a bottle ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 % Relative Humidity
- Air changes (per hr): not stated
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100, 50, 25 % (w/v)
No. of animals per dose:
4 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
Test item concentrations of 100 % (the undiluted test item itself) and 50 % in the vehicle (AOO) were tested. The applicability of the undiluted test item was acceptable. The 50 % concentration of test item in AOO was real solution and applicability of this formulation on the ears of animals was also acceptable.
- Irritation:
None.
- Lymph node proliferation response:
Not examined.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
Local Lymph Node Assay.

- Criteria used to consider a positive response:
The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was a liquid, hence the maximum concentration of 100 % (the undiluted test item) was tested. Solubility of the test item in Acetone: Olive oil 4:1 (v/v) mixture (AOO) was examined and found to be acceptable. Since AOO is the most preferred solvent according to OECD 429 guideline, no other solvents were examined. The 50 % and 25 % formulations of test item in AOO were real solutions. Applicability of the undiluted test item and the formulations on the ears of animals was acceptable.
Each mouse was topically treated with 25 µL of the appropriate formulation of 3 different concentrations of the test item, with the positive control substance (positive control groups) and with the vehicle (negative control group) using a pipette, on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Value:
31.8
Test group / Remarks:
100 %
Parameter:
SI
Value:
24.8
Test group / Remarks:
50 %
Parameter:
SI
Value:
23.4
Test group / Remarks:
25 %
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Please refer to the table "DPM, DPN and Stimulation Index Values for all Groups in the Main Test"
Cellular proliferation data / Observations:
Significant lymphoproliferative response (SI > 3) was noted for Sika Hardener MH at all concentrations tested. The stimulation index values were 31.8, 24.8 and 23.4 at concentrations of 100 %, 50 % and 25 %, respectively. The stimulation index values were compatible with a linear biological dose-dependent response.

DPM, DPN and Stimulation Index Values for all Groups in the Main Test

Test Group Measured DPM/Group  Group DPM  DPN (DPN/Node)  Stimulation Index Value 
Negative control 5088 5038.5 629.8 1.0
Positive control 25 % HCA in AOO 37158 37108.5 4638.6 7.4
SIKA Hardener MI 100 % 160071 160021 20002.7 31.8
SIKA Hardener MI 50 % 124943 124893.5 15611.7 24.8
SIKA Hardener MI 25 %  118078 118028.5 14753.6 23.4
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the results obtained from the test, SIKA Hardener MH was considered as skin sensitising in Cat. 1.
Executive summary:

The aim of this study was to determine the skin sensitization potential of Sika Hardener MH following dermal exposure in the Local Lymph Node Assay performed according to OECD 429, EU Method B.42 and OPPTS 870.2600. The test item is a liquid, hence, the maximum concentration of 100 % (the undiluted test item) was tested. Solubility of the test item in Acetone: Olive oil 4:1 (v/v) mixture (AOO) was examined and found to be acceptable. Since AOO is the most preferred solvent according to OECD 429 guideline, no other solvents were examined. The 50 % and 25 % formulations of test item in AOO were real solutions. Applicability of the undiluted test item and the formulations on the ears of animals was acceptable. A preliminary irritation/toxicity test was performed showing that application of test item up to 100 % was acceptable.

In the main assay 20 female CBA/Ca mice were allocated to five groups of four animals each:

-    three groups received Sika Hardener MH at concentrations of 100 %, 50 % or 25 %,

-    the negative control group received the vehicle (AOO),

-    the positive control groups receiveda-Hexylcinnamaldehyde (HCA) at concentration of 25 %.

Each substance was applied on the external surface of each ear (25 mL/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 local lymph nodes was determined by measuring incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

No mortality or significant systemic clinical signs were observed during the study. In the 100 % test item treated group twitching (intermittent) of the animals was observed approximately 1 hour after the third treatment on Day 3, but all animals were normal approximately 8 hours after the treatment and remained normal (i.e. no signs of systemic toxicity observed) during the subsequent days. No significant treatment related effects were observed on animal body weights in any treatment groups. No cutaneous reactions were observed at the site of the treatment (on the ears) in any of the treated groups. Slight, but not significant erythema was observed in the test item treated groups (presumably because of the slight accumulation of the viscous test item) at the base of ears of animals. Slight loss of hair (1/4 animals) was observed in the 100 % dose group. The effect was local (on the top of the head) and not extensive. The above mentioned effects were considered not to be significant and could not have effect on the proliferation results.

Stimulation index values of the test item were 31.8, 24.8 and 23.4 at treatment concentrations of 100 %, 50 % and 25 %, respectively. The stimulation index values were compatible with a biological dose-related response.

Normally, the EC3 value (the theoretical concentration of the test item in the test solution, leading a three fold increase of lymph node cell proliferation over the control) is estimated by linear interpolation using the reported SI values and the corresponding concentrations immediately above and below the SI value of 3. Since no SI value below 3 was observed with the test item, no estimation of the EC3 value was performed in this LLNA.

α-Hexylcinnamaldehyde, dissolved in AOO at concentration of 25 % (w/v) was used as positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response was noted for the positive control chemical with a stimulation index value of 7.4. The positive control results obtained confirmed the validity of the assay.

In conclusion, the test item SIKA Hardener MH was determined to be skin sensitizing in the LLNA assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

OECD 429

The aim of this study was to determine the skin sensitization potential of Sika Hardener MH following dermal exposure in the Local Lymph Node Assay performed according to OECD 429, EU Method B.42 and OPPTS 870.2600.The test item is a liquid, hence, the maximum concentration of 100 % (the undiluted test item) was tested. Solubility of the test item in Acetone: Olive oil 4:1 (v/v) mixture (AOO) was examined and found to be acceptable. Since AOO is the most preferred solvent according to OECD 429 guideline, no other solvents were examined. The 50 % and 25 % formulations of test item in AOO were real solutions. Applicability of the undiluted test item and the formulations on the ears of animals was acceptable. A preliminary irritation/toxicity test was performed showing that application of test item up to 100 % was acceptable.

In the main assay 20 female CBA/Ca mice were allocated to five groups of four animals each:

-    three groups received Sika Hardener MH at concentrations of 100 %, 50 % or 25 %,

-    the negative control group received the vehicle (AOO),

-    the positive control groups receiveda-Hexylcinnamaldehyde (HCA) at concentration of 25 %.

Each substance was applied on the external surface of each ear (25 mL/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 local lymph nodes was determined by measuring incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

No mortality or significant systemic clinical signs were observed during the study. In the 100 % test item treated group twitching (intermittent) of the animals was observed approximately 1 hour after the third treatment on Day 3, but all animals were normal approximately 8 hours after the treatment and remained normal (i.e. no signs of systemic toxicity observed) during the subsequent days. No significant treatment related effects were observed on animal body weights in any treatment groups. No cutaneous reactions were observed at the site of the treatment (on the ears) in any of the treated groups. Slight, but not significant erythema was observed in the test item treated groups (presumably because of the slight accumulation of the viscous test item) at the base of ears of animals. Slight loss of hair (1/4 animals) was observed in the 100 % dose group. The effect was local (on the top of the head) and not extensive. The above mentioned effects were considered not to be significant and could not have effect on the proliferation results.

Stimulation index values of the test item were 31.8, 24.8 and 23.4 at treatment concentrations of 100 %, 50 % and 25 %, respectively. The stimulation index values were compatible with a biological dose-related response.

Normally, the EC3 value (the theoretical concentration of the test item in the test solution, leading a three fold increase of lymph node cell proliferation over the control) is estimated by linear interpolation using the reported SI values and the corresponding concentrations immediately above and below the SI value of 3. Since no SI value below 3 was observed with the test item, no estimation of the EC3 value was performed in this LLNA.

α-Hexylcinnamaldehyde, dissolved in AOO at concentration of 25 % (w/v) was used as positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response was noted for the positive control chemical with a stimulation index value of 7.4. The positive control results obtained confirmed the validity of the assay.

In conclusion, the test item SIKA Hardener MH was determined to be skin sensitizing in the LLNA assay.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No. 1272/2008. As a result the substance is considered to be classified as a skin sensitiser in Cat. 1 (H317) under Regulation (EC) No. 1272/2008, as amended for the twelfth time in Regulation (EU) No. 2019/521.