Dodecanoic acid, 3-[[2-(2-hydroxyethoxy)ethyl]imino]-2,2-dimethylpropyl ester

Endpoint summary

Description of key information

Sika Hardener LG was assessed in a Mouse Local Lymphnode Assay (LLNA, EU method B.42). Based on the results obtained, Sika Hardener LG was considered to have a weak sensitisation potential (weak-sensitiser) in the LLNA.

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:

2008-09-10 to 2008-10-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: 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.

Vehicle:

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

Concentration:

25; 50; 100 % (w/v) (25 µL)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
A preliminary irritationltoxicity test was performed with the test item at concentrations of 100 % (the undiluted test item) and 50 % (%w/v) in the selected vehicle. The applicability and biocompatibility of the test item on the ears of animals at the maximum concentration of test item of 100 % was acceptable.

MAIN STUDY
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 test item 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 and the obtained values were used to calculate stimulation indices (SI).

Proliferation Assay
Injection of tritiated thymidine:
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 p1 of sterile PBS (phosphate buffered saline) containing 20 pCi (approximately) of 'HTdR using a gauge 25Gx1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours.

Removal and preparation of draining auricural lymph nodes:
Five hours after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group were pooled and collected in a Petri dish containing small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of single cell suspension of lymph node cells:
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 OC. After centrifugation each supernatant was removed leaving 1-2 mL supernatant above each pellet. Pellets were gently agitated before making up to 10 ml with PBS and resuspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of incorporated 3HTdR:
After the final wash each supernatant was removed leaving a small volume (<0.5 mL) of supernatant above each pellet. Each pellet was gently agitated before suspending the LNCs in 3 ml of 5 % TCA (trichloroacetic acid) for precipitation of macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approximately 18 hours) each precipitate was recovered by centrifugation at 190 x g for 10 minutes, supernatants were removed and pellets were suspended in 1 mL of 5 % TCA and dispersed using ultrasonic water bath. Each precipitate was transferred to a suitable sized scintillation vial with 10 mL of scintillation liquid and thoroughly mixed. The vials were
loaded to a scintillation counter and 3HTdR incorporatiodn was measured for up to 10 minutes per sample. The counter expressed the 3HtdR incorporation as the number of radioactive disintegrations per minute (DPM).
Similarly, background 3HTdR levles wered also measured in two 1 mL aliquots of 5 % TCA.

Interpretation of results:
The test item is regarded as a sensitiser if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- 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)

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.

Key result

Parameter:

SI

Value:

5

Test group / Remarks:

test item (100%)

Key result

Parameter:

SI

Value:

2.6

Test group / Remarks:

test item (50%)

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

test item (25%)

Key result

Parameter:

EC3

Value:

58.3

Test group / Remarks:

test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
A significant lymphoproliferative response was noted for the test item at a concentration of 100 %. The stimulation index values were 5.0, 2.6 and 1.4 at concentrations of 100 % (undiluted test item), 50 % and 25 %, respectively. The stimulation index values were compatible with the conventional biological dose-response.

EC3 CALCULATION
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) was estimated by linear interpolation using the reported SI values and the corresponding concentrations immediately above and below the SI value of 3. The estimated EC3 value of the test item was 58.3 %.

CLINICAL OBSERVATIONS & BODY WEIGHTS:
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.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the conditions of the present assay Sika Hardener LG, tested as supplied and in a suitable vehicle, was shown to have sensitisation potential in the Local Lymph Node Assay.

Executive summary:

A LLNA test was conducted with Sika Hardener LG. 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. 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) was estimated by linear interpolation using the reported SI values and the corresponding concentrations immediately above and below the SI value of 3. The estimated EC3 value of Sika Hardener LG was 58.3 % in this LLNA. On the basis of published classification of contact allergens according to their potency Sika Hardener LG can be ranked among weak sensitisers (100 >= EC3 (%) >=10). Under the conditions of the present assay Sika Hardener LG was shown to have weak sensitisation potential (weak-sensitiser) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A LLNA test was conducted with Sika Hardener LG according to EU method B.42 and OECD guideline no.429. No mortality or signs of systemic toxicity were observed during the study. No significant effects were observed in animal body weights. No irritation or other cutaneous effects were observed in any of the groups treated. The LLNA EC3 value was 58.3 %. On the basis of published classification of contact allergens according to their potency Sika Hardener LG was ranked among weak sensitisers (100 ≥ EC3 (%) ≥10).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the presented LLNA study, Sika Hardener LG was classified as a sensitiser Cat. 1B (H317: May cause an allergic skin reaction), according to according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.